Compositions Containing, Methods Involving, and Uses of Non-Natural Amino Acids and Polypeptides

ABSTRACT

Disclosed herein are non-natural amino acids and polypeptides that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of possible functionalities, but typical have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such polypeptides. Typically, the modified non-natural amino acid polypeptides include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, including therapeutic, diagnostic, and other biotechnology uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No.60/638,418, filed on Dec. 22, 2004, U.S. Provisional Application No.60/638,527, filed on Dec. 22, 2004, U.S. Provisional Application No.60/639,195, filed on Dec. 22, 2004, U.S. Provisional Application No.60/696,210, filed on Jul. 1, 2005, U.S. Provisional Application No.60/696,302, filed on Jul. 1, 2005, and U.S. Provisional Application No.60/696,068, filed on Jul. 1, 2005, the disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The ability to incorporate non-genetically encoded amino acids (i.e.,“non-natural amino acids”) into proteins permits the introduction ofchemical functional groups that could provide valuable alternatives tothe naturally-occurring functional groups, such as the epsilon —NH₂ oflysine, the sulfhydryl —SH of cysteine, the imino group of histidine,etc. Certain chemical functional groups are known to be inert to thefunctional groups found in the 20 common, genetically-encoded aminoacids but react cleanly and efficiently to form stable linkages withfunctional groups that can be incorporated onto non-natural amino acids.

Methods are now available to selectively introduce chemical functionalgroups that are not found in proteins, that are chemically inert to allof the functional groups found in the 20 common, genetically-encodedamino acids and that may be used to react efficiently and selectivelywith reagents comprising certain functional groups to form stablecovalent linkages.

SUMMARY OF THE INVENTION

Described herein are methods, compositions, techniques and strategiesfor making, purifying, characterizing, and using non-natural aminoacids, non-natural amino acid polypeptides and modified non-naturalamino acid polypeptides. In one aspect are methods, compositions,techniques and strategies for derivatizing a non-natural amino acidand/or a non-natural amino acid polypeptide. In one embodiment, suchmethods, compositions, techniques and strategies involved chemicalderivatization, in other embodiments, biological derivatization, inother embodiments, physical derivatization, in other embodiments acombination of derivatizations. In further or additional embodiments,such derivatizations are regioselective. In further or additionalembodiments, such derivatizations are regiospecific. In further oradditional embodiments, such derivatizations are rapid at ambienttemperature. In further or additional embodiments, such derivatizationsoccur in aqueous solutions. In further or additional embodiments, suchderivatizations occur at a pH between about 4 and about 10. In furtheror additional embodiments, with the addition of an accelerant suchderivations are stoichiometric, near stoichiometric orstoichiometric-like in both the non-natural amino acid containingreagent and the derivatizing reagent. In further or additionalembodiments are provided methods which, with the addition of anaccelerant, allow the stoichiometric, near stoichiometric orstoichiometric-like incorporation of a desired group onto a non-naturalamino acid polypeptide. In further or additional embodiments areprovided strategies, reaction mixtures, synthetic conditions which, withthe addition of an accelerant, allow the stoichiometric, nearstoichiometric or stoichiometric-like incorporation of a desired grouponto a non-natural amino acid polypeptide.

In one aspect are non-natural amino acids for the chemicalderivatization of peptides and proteins based upon an oxime linkage. Infurther or additional embodiments, the non-natural amino acid isincorporated into a polypeptide, that is, such embodiments arenon-natural amino acid polypeptides. In further or additionalembodiments, the non-natural amino acids are functionalized on theirsidechains such that their reaction with a derivatizing moleculegenerates an oxime linkage. In further or additional embodiments arenon-natural amino acid polypeptides that can react with a derivatizingmolecule to generate an oxime-containing non-natural amino acidpolypeptide. In further or additional embodiments, the non-natural aminoacids are selected from amino acids having carbonyl, dicarbonyl, acetal,hydroxylamine, or oxime sidechains. In further or additionalembodiments, the non-natural amino acids are selected from amino acidshaving protected or masked carbonyl, dicarbonyl, hydroxylamine, or oximesidechains. In further or additional embodiments, the non-natural aminoacids comprise an oxime-masked sidechain. In further or additionalembodiments, the non-natural amino acids comprise carbonyl or dicarbonylsidechains where the carbonyl or dicarbonyl is selected from a ketone oran aldehyde. In another embodiment are non-natural amino acidscontaining a functional group that is capable of forming an oxime upontreatment with an appropriately functionalized co-reactant. In a furtheror additional embodiment, the non-natural amino acids resemble a naturalamino acid in structure but contain one of the aforementioned functionalgroups. In another or further embodiment the non-natural amino acidsresemble phenylalanine or tyrosine (aromatic amino acids); while in aseparate embodiment, the non-natural amino acids resemble alanine andleucine (hydrophobic amino acids). In one embodiment, the non-naturalamino acids have properties that are distinct from those of the naturalamino acids. In one embodiment, such distinct properties are thechemical reactivity of the sidechain, in a further embodiment thisdistinct chemical reactivity permits the sidechain of the non-naturalamino acid to undergo a reaction while being a unit of a polypeptideeven though the sidechains of the naturally-occurring amino acid unitsin the same polypeptide do not undergo the aforementioned reaction. In afurther embodiment, the sidechain of the non-natural amino acid has achemistry orthogonal to those of the naturally-occurring amino acids. Ina further embodiment, the sidechain of the non-natural amino acidcomprises an electrophile-containing moiety; in a further embodiment,the electrophile-containing moiety on the sidechain of the non-naturalamino acid can undergo nucleophilic attack to generate anoxime-derivatized protein. In any of the aforementioned embodiments inthis paragraph, the non-natural amino acid may exist as a separatemolecule or may be incorporated into a polypeptide of any length; if thelatter, then the polypeptide may further incorporate naturally-occurringor non-natural amino acids.

In another aspect are hydroxylamine-substituted molecules for theproduction of derivatized non-natural amino acid polypeptides based uponan oxime linkage. In a further embodiment are hydroxylamine-substitutedmolecules used to derivatize carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptides via the formation of an oximelinkage between the derivatizing molecule and the carbonyl- ordicarbonyl-containing non-natural amino acid polypeptide. In furtherembodiments the aforementioned carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptides are keto-containing non-naturalamino acid polypeptides. In further or additional embodiments, thecarbonyl- or dicarbonyl-containing non-natural amino acids comprisesidechains selected from a ketone or an aldehyde. In further oradditional embodiments, the hydroxylamine-substituted molecules comprisea group selected from: a label; a dye; a polymer; a water-solublepolymer; a derivative of polyethylene glycol; a photocrosslinker; acytotoxic compound; a drug; an affinity label; a photoaffinity label; areactive compound; a resin; a second protein or polypeptide orpolypeptide analog; an antibody or antibody fragment; a metal chelator;a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; aRNA; an antisense polynucleotide; a saccharide, a water-solubledendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label;a fluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety, a ligand, a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof. In further or additional embodiments, thehydroxylamine-substituted molecules are hydroxylamine-substitutedpolyethylene glycol (PEG) molecules. In a further embodiment, thesidechain of the non-natural amino acid has a chemistry orthogonal tothose of the naturally-occurring amino acids that allows the non-naturalamino acid to react selectively with the hydroxylamine-substitutedmolecules. In a further embodiment, the sidechain of the non-naturalamino acid comprises an electrophile-containing moiety that reactsselectively with the hydroxylamine-containing molecule; in a furtherembodiment, the electrophile-containing moiety on the sidechain of thenon-natural amino acid can undergo nucleophilic attack to generate anoxime-derivatized protein. In a further aspect related to theembodiments described in this paragraph are the modified non-naturalamino acid polypeptides that result from the reaction of thederivatizing molecule with the non-natural amino acid polypeptides.Further embodiments include any further modifications of the alreadymodified non-natural amino acid polypeptides.

In another aspect are carbonyl- or dicarbonyl-substituted molecules forthe production of derivatized non-natural amino acid polypeptides basedupon an oxime linkage. In a further embodiment are carbonyl- ordicarbonyl-substituted molecules used to derivatize oxime-containingnon-natural amino acid polypeptides via an oxime exchange reactionbetween the derivatizing molecule and the oxime-containing peptide orprotein. In a further embodiment are carbonyl- or dicarbonyl-substitutedmolecules that can undergo oxime exchange with an oxime-containingnon-natural amino acid polypeptide in a pH range between about 4 andabout 8. In a further embodiment are carbonyl- or dicarbonyl-substitutedmolecules used to derivatize oxime-containing orhydroxylamine-containing non-natural amino acid polypeptides via theformation of an oxime linkage between the derivatizing molecule and theoxime-containing (thus forming a new oxime linkage via an exchange-typereaction) or hydroxylamine-containing non-natural amino acidpolypeptides. In a further embodiment the carbonyl- ordicarbonyl-substituted molecules are aldehyde substituted molecules. Infurther embodiments, the carbonyl- or dicarbonyl-substituted moleculescomprise a group selected from: a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety, a ligand, a photoisomerizablemoiety; biotin; a biotin analogue; a moiety incorporating a heavy atom;a chemically cleavable group; a photocleavable group; an elongated sidechain; a carbon-linked sugar; a redox-active agent; an amino thioacid; atoxic moiety; an isotopically labeled moiety; a biophysical probe; aphosphorescent group; a chemiluminescent group; an electron dense group;a magnetic group; an intercalating group; a chromophore; an energytransfer agent; a biologically active agent; a detectable label; a smallmolecule; an inhibitory ribonucleic acid, a radionucleotide; aneutron-capture agent; a derivative of biotin; quantum dot(s); ananotransmitter; a radiotransmitter; an abzyme, an activated complexactivator, a virus, an adjuvant, an aglycan, an allergan, anangiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, asaponin, a shuttle vector, a macromolecule, a mimotope, a receptor, areverse micelle, and any combination thereof. In further or additionalembodiments, the aldehyde-substituted molecules are aldehyde-substitutedpolyethylene glycol (PEG) molecules. In a further embodiment, thesidechain of the non-natural amino acid has a chemistry orthogonal tothose of the naturally-occurring amino acids that allows the non-naturalamino acid to react selectively with the carbonyl- ordicarbonyl-substituted molecules. In a further embodiment, the sidechainof the non-natural amino acid comprises a moiety, by way of example anoxime or hydroxylamine group, which reacts selectively with thecarbonyl- or dicarbonyl-containing molecule; in a further embodiment,the nucleophilic moiety on the sidechain of the non-natural amino acidcan undergo electrophilic attack to generate an oxime-derivatizedprotein. In a further aspect related to the embodiments described inthis paragraph are the modified non-natural amino acid polypeptides thatresult from the reaction of the derivatizing molecule with thenon-natural amino acid polypeptides. Further embodiments include anyfurther modifications of the already modified non-natural amino acidpolypeptides.

In another aspect are mono-, bi- and multi-functional linkers for thegeneration of derivatized non-natural amino acid polypeptides based uponan oxime linkage. In one embodiment are molecular linkers (bi- andmulti-functional) that can be used to connect carbonyl- ordicarbonyl-containing non-natural amino acid polypeptides to othermolecules. In another embodiment are molecular linkers (bi- andmulti-functional) that can be used to connect oxime- orhydroxylamine-containing non-natural amino acid polypeptides to othermolecules. In another embodiment the carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptides comprise a ketone and/or an aldehydesidechain. In an embodiment utilizing an oxime- orhydroxylamine-containing non-natural amino acid polypeptide, themolecular linker contains a carbonyl or dicarbonyl group at one of itstermini; in further embodiments, the carbonyl or dicarbonyl group isselected from an aldehyde group or a ketone group. In further oradditional embodiments, the hydroxylamine-substituted linker moleculesare hydroxylamine-substituted polyethylene glycol (PEG) linkermolecules. In further or additional embodiments, the carbonyl- ordicarbonyl-substituted linker molecules are carbonyl- ordicarbonyl-substituted polyethylene glycol (PEG) linker molecules. Infurther embodiments, the phrase “other molecules” includes, by way ofexample only, proteins, other polymers and small molecules. In furtheror additional embodiments, the hydroxylamine-containing molecularlinkers comprise the same or equivalent groups on all termini so thatupon reaction with a carbonyl- or dicarbonyl-containing non-naturalamino acid polypeptide, the resulting product is thehomo-multimerization of the carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptide. In further embodiments, thehomo-multimerization is a homo-dimerization. In further or additionalembodiments, the carbonyl- or dicarbonyl-containing molecular linkerscomprise the same or equivalent groups on all termini so that uponreaction with an oxime- or hydroxylamine-containing non-natural aminoacid polypeptide, the resulting product is the homo-multimerization ofthe oxime- or hydroxylamine-containing non-natural amino acidpolypeptide. In further embodiments, the homo-multimerization is ahomo-dimerization. In a further embodiment, the sidechain of thenon-natural amino acid has a chemistry orthogonal to those of thenaturally-occurring amino acids that allows the non-natural amino acidto react selectively with the hydroxylamine-substituted linkermolecules. In a further embodiment, the sidechain of the non-naturalamino acid has a chemistry orthogonal to those of thenaturally-occurring amino acids that allows the non-natural amino acidto react selectively with the carbonyl- or dicarbonyl-substituted linkermolecules. In a further embodiment, the sidechain of the non-naturalamino acid comprises an electrophile-containing moiety that reactsselectively with the hydroxylamine-containing linker molecule; in afurther embodiment, the electrophile-containing moiety on the sidechainof the non-natural amino acid can undergo nucleophilic attack by thehydroxylamine-containing linker molecule to generate anoxime-derivatized protein. In a further aspect related to theembodiments described in this paragraph are the linked “modified orunmodified” non-natural amino acid polypeptides that result from thereaction of the linker molecule with the non-natural amino acidpolypeptides. Further embodiments include any further modifications ofthe already linked “modified or unmodified” non-natural amino acidpolypeptides.

In one aspect are methods to derivatize proteins via the condensation ofcarbonyl or dicarbonyl and hydroxylamine reactants to generate anoxime-based product. Included within this aspect are methods for thederivatization of proteins based upon the condensation of carbonyl- ordicarbonyl- and hydroxylamine-containing reactants to generate anoxime-derivatized protein adduct. In additional or further embodimentsare methods to derivatize keto-containing proteins withhydroxylamine-functionalized polyethylene glycol (PEG) molecules. In yetadditional or further aspects are methods to derivatize oxime-containingproteins via an oxime exchange reaction between a carbonyl- ordicarbonyl-containing derivatizing molecule and the oxime-containingpeptide or protein. In yet additional or further aspects, thehydroxylamine-substituted molecule can include proteins, other polymers,and small molecules.

In another aspect are methods for the chemical synthesis ofhydroxylamine-substituted molecules for the derivatization ofketo-substituted proteins. In another aspect are methods for thechemical synthesis of hydroxylamine-substituted molecules for thederivatization of aldehyde-substituted proteins. In one embodiment, thehydroxylamine-substituted molecule can comprise peptides, other polymers(non-branched and branched) and small molecules. In one embodiment aremethods for the preparation of hydroxylamine-substituted moleculessuitable for the derivatization of carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptides, including by way of example only,keto-containing non-natural amino acid polypeptides. In a further oradditional embodiment, the non-natural amino acids are incorporatedsite-specifically during the in vivo translation of proteins. In afurther or additional embodiment, the hydroxylamine-substitutedmolecules allow for the site-specific derivatization of this carbonyl-or dicarbonyl-containing non-natural amino acid via nucleophilic attackof the carbonyl or dicarbonyl group to form an oxime-derivatizedpolypeptide in a site-specific fashion. In a further or additionalembodiment, the method for the preparation of hydroxylamine-substitutedmolecules provides access to a wide variety of site-specificallyderivatized polypeptides. In a further or additional embodiment aremethods for synthesizing hydroxylamine-functionalized polyethyleneglycol (PEG) molecules.

In another aspect are methods for the chemical synthesis of carbonyl- ordicarbonyl-substituted molecules for the derivatization ofoxime-substituted non-natural amino acid polypeptides. In oneembodiment, the carbonyl- or dicarbonyl-substituted molecule is aketo-substituted molecule. In one embodiment, the carbonyl- ordicarbonyl-substituted molecule is an aldehyde-substituted molecule. Inanother embodiment, the carbonyl- or dicarbonyl-substituted moleculesinclude proteins, polymers (non-branched and branched) and smallmolecules. In a further or additional embodiment, such methodscomplement technology that enables the site-specific incorporation ofnon-natural amino acids during the in vivo translation of proteins. In afurther or additional embodiment are general methods for the preparationof carbonyl- or dicarbonyl-substituted molecules suitable for reactionwith oxime-containing non-natural amino acid polypeptides to providesite-specifically derivatized non-natural amino acid polypeptides. In afurther or additional embodiment are methods for synthesizing carbonyl-or dicarbonyl-substituted polyethylene glycol (PEG) molecules.

In another aspect are methods for the chemical derivatization ofcarbonyl- or dicarbonyl-substituted non-natural amino acid polypeptidesusing a hydroxylamine-containing bi-functional linker. In one embodimentare methods for attaching a hydroxylamine-substituted linker to acarbonyl- or dicarbonyl-substituted protein via a condensation reactionto generate an oxime linkage. In further or additional embodiments, thecarbonyl- or dicarbonyl-substituted non-natural amino acid is aketo-substituted non-natural amino acid. In further or additionalembodiments, the non-natural amino acid polypeptides are derivatizedsite-specifically and/or with precise control of three-dimensionalstructure, using a hydroxylamine-containing bi-functional linker. In oneembodiment, such methods are used to attach molecular linkers(including, but not limited to, mono- bi- and multi-functional linkers)to carbonyl- or dicarbonyl-containing (including, but not limited to,keto-containing and aldehyde-containing) non-natural amino acidpolypeptides, wherein at least one of the linker termini contains ahydroxylamine group which can link to the carbonyl- ordicarbonyl-containing non-natural amino acid polypeptides via an oximelinkage. In a further or additional embodiment, these linkers are usedto connect the carbonyl- or dicarbonyl-containing non-natural amino acidpolypeptides to other molecules, including by way of example, proteins,other polymers (branched and non-branched) and small molecules.

In some embodiments, the non-natural amino acid polypeptide is linked toa water soluble polymer. In some embodiments, the water soluble polymercomprises a polyethylene glycol moiety. In some embodiments, thepolyethylene glycol molecule is a bifunctional polymer. In someembodiments, the bifunctional polymer is linked to a second polypeptide.In some embodiments, the second polypeptide is identical to the firstpolypeptide, in other embodiments, the second polypeptide is a differentpolypeptide. In some embodiments, the non-natural amino acid polypeptidecomprises at least two amino acids linked to a water soluble polymercomprising a poly(ethylene glycol) moiety.

In some embodiments, the non-natural amino acid polypeptide comprises asubstitution, addition or deletion that increases affinity of thenon-natural amino acid polypeptide for a receptor. In some embodiments,the non-natural amino acid polypeptide comprises a substitution,addition, or deletion that increases the stability of the non-naturalamino acid polypeptide. In some embodiments, the non-natural amino acidpolypeptide comprises a substitution, addition, or deletion thatincreases the aqueous solubility of the non-natural amino acidpolypeptide. In some embodiments, the non-natural amino acid polypeptidecomprises a substitution, addition, or deletion that increases thesolubility of the non-natural amino acid polypeptide produced in a hostcell. In some embodiments, the non-natural amino acid polypeptidecomprises a substitution, addition, or deletion that modulates proteaseresistance, serum half-life, immunogenicity, and/or expression relativeto the amino-acid polypeptide without the substitution, addition ordeletion.

In some embodiments, the non-natural amino acid polypeptide is anagonist, partial agonist, antagonist, partial antagonist, or inverseagonist. In some embodiments, the agonist, partial agonist, antagonist,partial antagonist, or inverse agonist comprises a non-natural aminoacid linked to a water soluble polymer. In some embodiments, the waterpolymer comprises a polyethylene glycol moiety. In some embodiments, thepolypeptide comprising a non-natural amino acid linked to a watersoluble polymer, for example, may prevent dimerization of thecorresponding receptor. In some embodiments, the polypeptide comprisinga non-natural amino acid linked to a water soluble polymer modulatesbinding of the polypeptide to a binding partner, ligand or receptor. Insome embodiments, the polypeptide comprising a non-natural amino acidlinked to a water soluble polymer modulates one or more properties oractivities of the polypeptide.

In some embodiments, the selector codon is selected from the groupconsisting of an amber codon, ochre codon, opal codon, a unique codon, arare codon, an unnatural codon, a five-base codon, and a four-basecodon.

Also described herein are methods of making a non-natural amino acidpolypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated polypeptide comprising anon-natural amino acid with a water soluble polymer comprising a moietythat reacts with the non-natural amino acid. In some embodiments, thenon-natural amino acid incorporated into is reactive toward a watersoluble polymer that is otherwise unreactive toward any of the 20 commonamino acids. In some embodiments, the water polymer comprises apolyethylene glycol moiety. The molecular weight of the polymer may beof a wide range, including but not limited to, between about 100 Da andabout 100,000 Da or more. The molecular weight of the polymer may bebetween about 100 Da and about 100,000 Da, including but not limited to,100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da,70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da. In some embodiments, the polyethylene glycol moleculeis a branched polymer. The molecular weight of the branched chain PEGmay be between about 1,000 Da and about 100,000 Da, including but notlimited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and 50,000Da. In some embodiments, the molecular weight of the branched chain PEGis between about 1,000 Da and 40,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 5,000 Da and40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and 20,000 Da.

Also described herein are compositions comprising a polypeptidecomprising at least one of the non-natural amino acids described hereinand a pharmaceutically acceptable carrier. In some embodiments, thenon-natural amino acid is linked to a water soluble polymer. Alsodescribed herein are pharmaceutical compositions comprising apharmaceutically acceptable carrier and a polypeptide, wherein at leastone amino acid is substituted by a non-natural amino acid. In someembodiments, the non-natural amino acid comprises a saccharide moiety.In some embodiments, the water soluble polymer is linked to thepolypeptide via a saccharide moiety. Also described herein are prodrugsof the non-natural amino acids, non-natural amino acid polypeptides, andmodified non-natural amino acid polypeptides; further described hereinare compositions comprising such prodrugs and a pharmaceuticallyacceptable carrier. Also described herein are metabolites of thenon-natural amino acids, non-natural amino acid polypeptides, andmodified non-natural amino acid polypeptides; such metabolites may havea desired activity that complements or synergizes with the activity ofthe non-natural amino acids, non-natural amino acid polypeptides, andmodified non-natural amino acid polypeptides. Also described herein arethe use of the non-natural amino acids, non-natural amino acidpolypeptides, and modified non-natural amino acid polypeptides describedherein to provide a desired metabolite to an organism, including apatient in need of such metabolite.

Also described herein are cells comprising a polynucleotide encoding thepolypeptide comprising a selector codon. In some embodiments, the cellscomprise an orthogonal RNA synthetase and/or an orthogonal tRNA forsubstituting a non-natural amino acid into the polypeptide. In someembodiments the cells are in a cell culture, whereas in otherembodiments the cells of part of a multicellular organism, includingamphibians, reptiles, birds, and mammals. In any of the cellembodiments, further embodiments include expression of thepolynucleotide to produce the non-natural amino acid polypeptide. Inother embodiments are organisms that can utilize the non-natural aminoacids described herein to produce a non-natural amino acid polypeptide,including a modified non-natural amino acid polypeptide. In otherembodiments are organisms containing the non-natural amino acids, thenon-natural amino acid polypeptides, and/or the modified non-naturalamino acid polypeptides described herein. Such organisms includeunicellular and multicellular organisms, including amphibians, reptiles,birds, and mammals. In some embodiments, the non-natural amino acidpolypeptide is produced in vitro. In some embodiments, the non-naturalamino acid polypeptide is produced in cell lysate. In some embodiments,the non-natural amino acid polypeptide is produced by ribosomaltranslation.

Also described herein are methods of making a polypeptide comprising anon-natural amino acid. In some embodiments, the methods compriseculturing cells comprising a polynucleotide or polynucleotides encodinga polypeptide, an orthogonal RNA synthetase and/or an orthogonal tRNAunder conditions to permit expression of the polypeptide; and purifyingthe polypeptide from the cells and/or culture medium.

Also described herein are libraries of the non-natural amino acidsdescribed herein or libraries of the non-natural amino acid polypeptidesdescribed herein, or libraries of the modified non-natural amino acidpolypeptides described herein, or combination libraries thereof. Alsodescribed herein are arrays containing at least one non-natural aminoacid, at least one non-natural amino acid polypeptide, and/or at leastone modified non-natural amino acid. Also described herein are arrayscontaining at least one polynucleotide encoding a polypeptide comprisinga selector codon. The arrays described herein may be used to screen forthe production of the non-natural amino acid polypeptides in an organism(either by detecting transcription of the polynucleotide encoding thepolypeptide or by detecting the translation of the polypeptide).

Also described herein are methods for screening libraries describedherein for a desired activity, or for using the arrays described hereinto screen the libraries described herein, or for other libraries ofcompounds and/or polypeptides and/or polynucleotides for a desiredactivity. Also described herein is the use of such activity data fromlibrary screening to develop and discover new therapeutic agents, aswell as the therapeutic agents themselves.

Also described herein are methods of increasing therapeutic half-life,serum half-life or circulation time of a polypeptide. In someembodiments, the methods comprise substituting at least one non-naturalamino acid for any one or more amino acids in a naturally occurringpolypeptide and/or coupling the polypeptide to a water soluble polymer.

Also described herein are methods of treating a patient in need of suchtreatment with an effective amount of a pharmaceutical composition whichcomprises a polypeptide comprising a non-natural amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-natural amino acid is coupled to a water soluble polymer.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one non-natural amino acid selected from the groupconsisting of an oxime-containing non-natural amino acid, acarbonyl-containing non-natural amino acid, a dicarbonyl-containingnon-natural amino acid, and a hydroxylamine-containing non-natural aminoacid. In other embodiments such non-natural amino acids have beenbiosynthetically incorporated into the polypeptide as described herein.In further or alternative embodiments such non-natural amino acidpolypeptide comprise at least one non-natural amino acid selected fromamino acids of Formula I-XVIII, XXX-XXXIV(A&B), or XXXX-XXXXIII.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide increases the bioavailability of the polypeptide relative tothe homologous naturally-occurring amino acid polypeptide.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide increases the safety profile of the polypeptide relative tothe homologous naturally-occurring amino acid polypeptide.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide increases the water solubility of the polypeptide relativeto the homologous naturally-occurring amino acid polypeptide.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide increases the therapeutic half-life of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.

The In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide increases the serum half-life of the polypeptide relative tothe homologous naturally-occurring amino acid polypeptide.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide extends the circulation time of the polypeptide relative tothe homologous naturally-occurring amino acid polypeptide.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide modulates the biological activity of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.

In further or alternative embodiments are methods for treating adisorder, condition or disease, the method comprising administering atherapeutically effective amount of a non-natural amino acid polypeptidecomprising at least one oxime-containing non-natural amino acid and theresulting biosynthetic oxime-containing non-natural amino acidpolypeptide modulates the immunogenicity of the polypeptide relative tothe homologous naturally-occurring amino acid polypeptide.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods and compositions described herein,which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions described herein belong. Although anymethods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the inventionsdescribed herein, the preferred methods, devices and materials are nowdescribed.

All publications and patents mentioned herein are incorporated herein byreference in their entirety for the purpose of describing anddisclosing, for example, the constructs and methodologies that aredescribed in the publications, which might be used in connection withthe presently described inventions. The publications discussed hereinare provided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors described herein are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason.

The term “affinity label,” as used herein, refers to a label whichreversibly or irreversibly binds another molecule, either to modify it,destroy it, or form a compound with it. By way of example, affinitylabels include, enzymes and their substrates, or antibodies and theirantigens.

The terms “alkoxy,” “alkylamino” and “alkylthio” are used in theirconventional sense, and refer to alkyl groups linked to molecules via anoxygen atom, an amino group, a sulfur atom, respectively.

The term “alkyl,” by itself or as part of another molecule, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C1-C10means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail herein, such as “heteroalkyl”, “haloalkyl” and“homoalkyl”.

The term “alkylene” by itself or as part of another molecule means adivalent radical derived from an alkane, as exemplified by (—CH2-)n,wherein n may be 1 to about 24. By way of example only, such groupsinclude, but are not limited to, groups having 10 or fewer carbon atomssuch as the structures —CH2CH2- and —CH2CH2CH2CH2-. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms. The term “alkylene,” unlessotherwise noted, is also meant to include those groups described hereinas “heteroalkylene.”

The term “amino acid” refers to naturally occurring and non-naturalamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally encoded amino acids are the 20 common amino acids (alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline) and pyrolysine and selenocysteine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, by way of example only, an α-carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group. Suchanalogs may have modified R groups (by way of example, norleucine) ormay have modified peptide backbones, while still retaining the samebasic chemical structure as a naturally occurring amino acid.Non-limiting examples of amino acid analogs include homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium.

Amino acids may be referred to herein by either their name, theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission.Additionally, nucleotides, may be referred to by their commonly acceptedsingle-letter codes.

An “amino terminus modification group” refers to any molecule that canbe attached to a terminal amine group. By way of example, such terminalamine groups may be at the end of polymeric molecules, wherein suchpolymeric molecules include, but are not limited to, polypeptides,polynucleotides, and polysaccharides. Terminus modification groupsinclude but are not limited to, various water soluble polymers, peptidesor proteins. By way of example only, terminus modification groupsinclude polyethylene glycol or serum albumin. Terminus modificationgroups may be used to modify therapeutic characteristics of thepolymeric molecule, including but not limited to increasing the serumhalf-life of peptides.

By “antibody fragment” is meant any form of an antibody other than thefull-length form. Antibody fragments herein include antibodies that aresmaller components that exist within full-length antibodies, andantibodies that have been engineered. Antibody fragments include but arenot limited to Fv, Fc, Fab, and (Fab′)2, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, heavy chains, light chains, and variableregions, and alternative scaffold non-antibody molecules, bispecificantibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed.Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Anotherfunctional substructure is a single chain Fv (scFv), comprised of thevariable regions of the immunoglobulin heavy and light chain, covalentlyconnected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56,3055-3061). These small (Mr 25,000) proteins generally retainspecificity and affinity for antigen in a single polypeptide and canprovide a convenient building block for larger, antigen-specificmolecules. Unless specifically noted otherwise, statements and claimsthat use the term “antibody” or “antibodies” specifically includes“antibody fragment” and “antibody fragments.”

The term “aromatic” or “aryl”, as used herein, refers to a closed ringstructure which has at least one ring having a conjugated pi electronsystem and includes both carbocyclic aryl and heterocyclic aryl (or“heteroaryl” or “heteroaromatic”) groups. The carbocyclic orheterocyclic aromatic group may contain from 5 to 20 ring atoms. Theterm includes monocyclic rings linked covalently or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups. An aromatic group can be unsubstituted or substituted.Non-limiting examples of “aromatic” or “aryl”, groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthracenyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedherein.

For brevity, the term “aromatic” or “aryl” when used in combination withother terms (including but not limited to, aryloxy, arylthioxy, aralkyl)includes both aryl and heteroaryl rings as defined above. Thus, the term“aralkyl” or “alkaryl” is meant to include those radicals in which anaryl group is attached to an alkyl group (including but not limited to,benzyl, phenethyl, pyridylmethyl and the like) including those alkylgroups in which a carbon atom (including but not limited to, a methylenegroup) has been replaced by a heteroatom, by way of example only, by anoxygen atom. Examples of such aryl groups include, but are not limitedto, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and thelike.

The term “arylene”, as used herein, refers to a divalent aryl radical.Non-limiting examples of “arylene” include phenylene, pyridinylene,pyrimidinylene and thiophenylene. Substituents for arylene groups areselected from the group of acceptable substituents described herein.

A “bifunctional polymer”, also referred to as a “bifunctional linker”,refers to a polymer comprising two functional groups that are capable ofreacting specifically with other moieties to form covalent ornon-covalent linkages. Such moieties may include, but are not limitedto, the side groups on natural or non-natural amino acids or peptideswhich contain such natural or non-natural amino acids. By way of exampleonly, a bifunctional linker may have a functional group reactive with agroup on a first peptide, and another functional group which is reactivewith a group on a second peptide, whereby forming a conjugate thatincludes the first peptide, the bifunctional linker and the secondpeptide. Many procedures and linker molecules for attachment of variouscompounds to peptides are known. See, e.g., European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; and 4,569,789 which are incorporated by reference herein intheir entirety. A “multi-functional polymer” also referred to as a“multi-functional linker”, refers to a polymer comprising two or morefunctional groups that are capable of reacting with other moieties. Suchmoieties may include, but are not limited to, the side groups on naturalor non-natural amino acids or peptides which contain such natural ornon-natural amino acids. (including but not limited to, amino acid sidegroups) to form covalent or non-covalent linkages. A bi-functionalpolymer or multi-functional polymer may be any desired length ormolecular weight, and may be selected to provide a particular desiredspacing or conformation between one or more molecules linked to acompound and molecules it binds to or the compound.

The term “bioavailability,” as used herein, refers to the rate andextent to which a substance or its active moiety is delivered from apharmaceutical dosage form and becomes available at the site of actionor in the general circulation. Increases in bioavailability refers toincreasing the rate and extent a substance or its active moiety isdelivered from a pharmaceutical dosage form and becomes available at thesite of action or in the general circulation. By way of example, anincrease in bioavailability may be indicated as an increase inconcentration of the substance or its active moiety in the blood whencompared to other substances or active moieties. A non-limiting exampleof a method to evaluate increases in bioavailability is given inexamples 88-92. This method may be used for evaluating thebioavailability of any polypeptide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include butare not limited to any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the methods andcompositions described herein include, but are not limited to, drugs,prodrugs, radionuclides, imaging agents, polymers, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, microbially derived toxins, and the like.

By “modulating biological activity” is meant increasing or decreasingthe reactivity of a polypeptide, altering the selectivity of thepolypeptide, enhancing or decreasing the substrate selectivity of thepolypeptide. Analysis of modified biological activity can be performedby comparing the biological activity of the non-natural polypeptide tothat of the natural polypeptide.

The term “biomaterial,” as used herein, refers to a biologically-derivedmaterial, including but not limited to material obtained frombioreactors and/or from recombinant methods and techniques.

The term “biophysical probe,” as used herein, refers to probes which candetect or monitor structural changes in molecules. Such moleculesinclude, but are not limited to, proteins and the “biophysical probe”may be used to detect or monitor interaction of proteins with othermacromolecules. Examples of biophysical probes include, but are notlimited to, spin-labels, a fluorophores, and photoactivatible groups.

The term “biosynthetically,” as used herein, refers to any methodutilizing a translation system (cellular or non-cellular), including useof at least one of the following components: a polynucleotide, a codon,a tRNA, and a ribosome. By way of example, non-natural amino acids maybe “biosynthetically incorporated” into non-natural amino acidpolypeptides using the methods and techniques described in section VIII“In vivo generation of polypeptides comprising non-natural amino acids”,and in the non-limiting example 14. Additionally, the methods for theselection of useful non-natural amino acids which may be“biosynthetically incorporated” into non-natural amino acid polypeptidesare described in the non-limiting examples 15-16.

The term “biotin analogue,” or also referred to as “biotin mimic”, asused herein, is any molecule, other than biotin, which bind with highaffinity to avidin and/or streptavidin.

The term “carbonyl” as used herein refers to a group containing at amoiety selecting from the group consisting of —C(O)—, —S(O)—, —S(O)₂—,and —C(S)—, including, but not limited to, groups containing a least oneketone group, and/or at least one aldehyde groups, and/or at least oneester group, and/or at least one carboxylic acid group, and/or at leastone thioester group. Such carbonyl groups include ketones, aldehydes,carboxylic acids, esters, and thioesters. In addition, such groups maybe part of linear, branched, or cyclic molecules.

The term “carboxy terminus modification group” refers to any moleculethat can be attached to a terminal carboxy group. By way of example,such terminal carboxy groups may be at the end of polymeric molecules,wherein such polymeric molecules include, but are not limited to,polypeptides, polynucleotides, and polysaccharides. Terminusmodification groups include but are not limited to, various watersoluble polymers, peptides or proteins. By way of example only, terminusmodification groups include polyethylene glycol or serum albumin.Terminus modification groups may be used to modify therapeuticcharacteristics of the polymeric molecule, including but not limited toincreasing the serum half-life of peptides.

The term “chemically cleavable group,” also referred to as “chemicallylabile”, as used herein, refers to a group which breaks or cleaves uponexposure to acid, base, oxidizing agents, reducing agents, chemicalinititiators, or radical initiators.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example only, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H2O2) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The term “cofactor,” as used herein, refers to an atom or moleculeessential for the action of a large molecule. Cofactors include, but arenot limited to, inorganic ions, coenzymes, proteins, or some otherfactor necessary for the activity of enzymes. Examples include, heme inhemoglobin, magnesium in chlorophyll, and metal ions for proteins.

“Cofolding,” as used herein, refers to refolding processes, reactions,or methods which employ at least two molecules which interact with eachother and result in the transformation of unfolded or improperly foldedmolecules to properly folded molecules. By way of example only,“cofolding,” employ at least two polypeptides which interact with eachother and result in the transformation of unfolded or improperly foldedpolypeptides to native, properly folded polypeptides. Such polypeptidesmay contain natural amino acids and/or at least one non-natural aminoacid.

A “comparison window,” as used herein, refers a segment of any one ofcontiguous positions used to compare a sequence to a reference sequenceof the same number of contiguous positions after the two sequences areoptimally aligned. Such contiguous positions include, but are notlimited to a group consisting of from about 20 to about 600 sequentialunits, including about 50 to about 200 sequential units, and about 100to about 150 sequential units. By way of example only, such sequencesinclude polypeptides and polypeptides containing non-natural aminoacids, with the sequential units include, but are not limited to naturaland non-natural amino acids. In addition, by way of example only, suchsequences include polynucleotides with nucleotides being thecorresponding sequential units. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, including but not limited to, by the localhomology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443, by the search for similarity method of Pearson andLipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Ausubel et al., Current Protocols in MolecularBiology (1995 supplement)).

By way of example, an algorithm which may be used to determine percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The term “conservatively modified variants” applies to both natural andnon-natural amino acid and natural and non-natural nucleic acidsequences, and combinations thereof. With respect to particular nucleicacid sequences, “conservatively modified variants” refers to thosenatural and non-natural nucleic acids which encode identical oressentially identical natural and non-natural amino acid sequences, orwhere the natural and non-natural nucleic acid does not encode a naturaland non-natural amino acid sequence, to essentially identical sequences.By way of example, because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Thus by way of exampleevery natural or non-natural nucleic acid sequence herein which encodesa natural or non-natural polypeptide also describes every possiblesilent variation of the natural or non-natural nucleic acid. One ofskill will recognize that each codon in a natural or non-natural nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a natural and non-natural nucleic acid which encodes anatural and non-natural polypeptide is implicit in each describedsequence.

As to amino acid sequences, individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single natural and non-natural aminoacid or a small percentage of natural and non-natural amino acids in theencoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of a natural and non-natural amino acid witha chemically similar amino acid. Conservative substitution tablesproviding functionally similar natural amino acids are well known in theart. Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of themethods and compositions described herein.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins:Structures and Molecular Properties (W HFreeman & Co.; 2nd edition (December 1993)

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. The heteroatom may include, but is notlimited to, oxygen, nitrogen or sulfur. Examples of cycloalkyl include,but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1 (1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 piperazinyl,2-piperazinyl, and the like. Additionally, the term encompassesmulticyclic structures, including but not limited to, bicyclic andtricyclic ring structures. Similarly, the term “heterocycloalkylene” byitself or as part of another molecule means a divalent radical derivedfrom heterocycloalkyl, and the term “cycloalkylene” by itself or as partof another molecule means a divalent radical derived from cycloalkyl.

The term “cyclodextrin,” as used herein, refers to cyclic carbohydratesconsisting of at least six to eight glucose molecules in a ringformation. The outer part of the ring contains water soluble groups; atthe center of the ring is a relatively nonpolar cavity able toaccommodate small molecules.

The term “cytotoxic,” as used herein, refers to a compound which harmscells.

“Denaturing agent” or “denaturant,” as used herein, refers to anycompound or material which will cause a reversible unfolding of apolymer. By way of example only, “denaturing agent” or “denaturants,”may cause a reversible unfolding of a protein. The strength of adenaturing agent or denaturant will be determined both by the propertiesand the concentration of the particular denaturing agent or denaturant.By way of example, denaturing agents or denaturants include, but are notlimited to, chaotropes, detergents, organic, water miscible solvents,phospholipids, or a combination thereof. Non-limiting examples ofchaotropes include, but are not limited to, urea, guanidine, and sodiumthiocyanate. Non-limiting examples of detergents may include, but arenot limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Non-limiting examples of organic, water miscible solventsinclude, but are not limited to, acetonitrile, lower alkanols(especially C₂-C₄ alkanols such as ethanol or isopropanol), or loweralkandiols (C₂-C₄ alkandiols such as ethylene-glycol) may be used asdenaturants. Non-limiting examples of phospholipids include, but are notlimited to, naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

The term “detectable label,” as used herein, refers to a label which maybe observable using analytical techniques including, but not limited to,fluorescence, chemiluminescence, electron-spin resonance,ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclearmagnetic resonance, magnetic resonance, and electrochemical methods.

The term “dicarbonyl” as used herein refers to a group containing atleast two moieties selected from the group consisting of —C(O)—, —S(O)—,—S(O)₂—, and —C(S)—, including, but not limited to, 1,2-dicarbonylgroups, a 1,3-dicarbonyl groups, and 1,4-dicarbonyl groups, and groupscontaining a least one ketone group, and/or at least one aldehydegroups, and/or at least one ester group, and/or at least one carboxylicacid group, and/or at least one thioester group. Such dicarbonyl groupsinclude diketones, ketoaldehydes, ketoacids, ketoesters, andketothioesters. In addition, such groups may be part of linear,branched, or cyclic molecules. The two moieties in the dicarbonyl groupmay be the same or different, and may include substituents that wouldproduce, by way of example only, an ester, a ketone, an aldehyde, athioester, or an amide, at either of the two moieties.

The term “drug,” as used herein, refers to any substance used in theprevention, diagnosis, alleviation, treatment, or cure of a disease orcondition.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The term “effective amount,” as used herein, refers to a sufficientamount of an agent or a compound being administered which will relieveto some extent one or more of the symptoms of the disease or conditionbeing treated. The result can be reduction and/or alleviation of thesigns, symptoms, or causes of a disease, or any other desired alterationof a biological system. By way of example, an agent or a compound beingadministered includes, but is not limited to, a natural amino acidpolypeptide, non-natural amino acid polypeptide, modified natural aminoacid polypeptide, or modified non-amino acid polypeptide. Compositionscontaining such natural amino acid polypeptides, non-natural amino acidpolypeptides, modified natural amino acid polypeptides, or modifiednon-natural amino acid polypeptides can be administered forprophylactic, enhancing, and/or therapeutic treatments. An appropriate“effective” amount in any individual case may be determined usingtechniques, such as a dose escalation study.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitratecadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, 98.5%, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a moleculewhich can either donate or accept energy from another molecule. By wayof example only, fluorescence resonance energy transfer (FRET) is adipole-dipole coupling process by which the excited-state energy of afluorescence donor molecule is non-radiatively transferred to anunexcited acceptor molecule which then fluorescently emits the donatedenergy at a longer wavelength.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. By way of example, “enhancing”the effect of therapeutic agents refers to the ability to increase orprolong, either in potency or duration, the effect of therapeutic agentson during treatment of a disease, disorder or condition. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of a therapeutic agent in the treatmentof a disease, disorder or condition. When used in a patient, amountseffective for this use will depend on the severity and course of thedisease, disorder or condition, previous therapy, the patient's healthstatus and response to the drugs, and the judgment of the treatingphysician.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya, including but not limited to animals(including but not limited to, mammals, insects, reptiles, birds, etc.),ciliates, plants (including but not limited to, monocots, dicots, andalgae), fungi, yeasts, flagellates, microsporidia, and protists.

The term “fatty acid,” as used herein, refers to carboxylic acids withabout C6 or longer hydrocarbon side chain.

The term “fluorophore,” as used herein, refers to a molecule which uponexcitation emits photons and is thereby fluorescent.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety,” as used herein, refer to portions or unitsof a molecule at which chemical reactions occur. The terms are somewhatsynonymous in the chemical arts and are used herein to indicate theportions of molecules that perform some function or activity and arereactive with other molecules.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “haloacyl,” as used herein, refers to acyl groups which containhalogen moieties, including, but not limited to, —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like.

The term “haloalkyl,” as used herein, refers to alkyl groups whichcontain halogen moieties, including, but not limited to, —CF₃ and—CH₂CF₃ and the like.

The term “heteroalkyl,” as used herein, refers to straight or branchedchain, or cyclic hydrocarbon radicals, or combinations thereof,consisting of an alkyl group and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. In addition, up to two heteroatoms may beconsecutive, such as, by way of example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

The term “heteroalkylene,” as used herein, refers to a divalent radicalderived from heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, the same or different heteroatoms can also occupy either or bothof the chain termini (including but not limited to, alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and thelike). Still further, for alkylene and heteroalkylene linking groups, noorientation of the linking group is implied by the direction in whichthe formula of the linking group is written. By way of example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—.

The term “heteroaryl” or “heteroaromatic,” as used herein, refers toaryl groups which contain at least one heteroatom selected from N, O,and S; wherein the nitrogen and sulfur atoms may be optionally oxidized,and the nitrogen atom(s) may be optionally quaternized. Heteroarylgroups may be substituted or unsubstituted. A heteroaryl group may beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of heteroaryl groups include 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

The term “homoalkyl,” as used herein refers to alkyl groups which arehydrocarbon groups.

The term “identical,” as used herein, refers to two or more sequences orsubsequences which are the same. In addition, the term “substantiallyidentical,” as used herein, refers to two or more sequences which have apercentage of sequential units which are the same when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using comparison algorithms or by manualalignment and visual inspection. By way of example only, two or moresequences may be “substantially identical” if the sequential units areabout 60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. Suchpercentages to describe the “percent identity” of two or more sequences.The identity of a sequence can exist over a region that is at leastabout 75-100 sequential units in length, over a region that is about 50sequential units in length, or, where not specified, across the entiresequence. This definition also refers to the complement of a testsequence. By way of example only, two or more polypeptide sequences areidentical when the amino acid residues are the same, while two or morepolypeptide sequences are “substantially identical” if the amino acidresidues are about 60% identical, about 65% identical, about 70%identical, about 75% identical, about 80% identical, about 85%identical, about 90% identical, or about 95% identical over a specifiedregion. The identity can exist over a region that is at least about75-100 amino acids in length, over a region that is about 50 amino acidsin length, or, where not specified, across the entire sequence of apolypeptide sequence. In addition, by way of example only, two or morepolynucleotide sequences are identical when the nucleic acid residuesare the same, while two or more polynucleotide sequences are“substantially identical” if the nucleic acid residues are about 60%identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. The identitycan exist over a region that is at least about 75-100 nucleic acids inlength, over a region that is about 50 nucleic acids in length, or,where not specified, across the entire sequence of a polynucleotidesequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “immunogenicity,” as used herein, refers to an antibodyresponse to administration of a therapeutic drug. The immunogenicitytoward therapeutic non-natural amino acid polypeptides can be obtainedusing quantitative and qualitative assays for detection ofanti-non-natural amino acid polypeptides antibodies in biologicalfluids. Such assays include, but are not limited to, Radioimmunoassay(RIA), Enzyme-linked immunosorbent assay (ELISA), luminescentimmunoassay (LIA), and fluorescent immunoassay (FIA). Analysis ofimmunogenicity toward therapeutic non-natural amino acid polypeptidesinvolves comparing the antibody response upon administration oftherapeutic non-natural amino acid polypeptides to the antibody responseupon administration of therapeutic natural amino acid polypeptides.

The term “intercalating agent,” also referred to as “intercalatinggroup,” as used herein, refers to a chemical that can insert into theintramolecular space of a molecule or the intermolecular space betweenmolecules. By way of example only an intercalating agent or group may bea molecule which inserts into the stacked bases of the DNA double helix.

The term “isolated,” as used herein, refers to separating and removing acomponent of interest from components not of interest. Isolatedsubstances can be in either a dry or semi-dry state, or in solution,including but not limited to an aqueous solution. The isolated componentcan be in a homogeneous state or the isolated component can be a part ofa pharmaceutical composition that comprises additional pharmaceuticallyacceptable carriers and/or excipients. Purity and homogeneity may bedetermined using analytical chemistry techniques including, but notlimited to, polyacrylamide gel electrophoresis or high performanceliquid chromatography. In addition, when a component of interest isisolated and is the predominant species present in a preparation, thecomponent is described herein as substantially purified. The term“purified,” as used herein, refers to a component of interest which isat least 85% pure, at least 90% pure, at least 95% pure, at least 99% orgreater pure. By way of example only, nucleic acids or proteins are“isolated” when such nucleic acids or proteins are free of at least someof the cellular components with which it is associated in the naturalstate, or that the nucleic acid or protein has been concentrated to alevel greater than the concentration of its in vivo or in vitroproduction. Also, by way of example, a gene is isolated when separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest.

The term “label,” as used herein, refers to a substance which isincorporated into a compound and is readily detected, whereby itsphysical distribution may be detected and/or monitored.

The term “linkage,” as used herein to refer to bonds or chemical moietyformed from a chemical reaction between the functional group of a linkerand another molecule. Such bonds may include, but are not limited to,covalent linkages and non-covalent bonds, while such chemical moietiesmay include, but are not limited to, esters, carbonates, iminesphosphate esters, hydrazones, acetals, orthoesters, peptide linkages,and oligonucleotide linkages. Hydrolytically stable linkages means thatthe linkages are substantially stable in water and do not react withwater at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages means thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meansthat the linkage can be degraded by one or more enzymes. By way ofexample only, PEG and related polymers may include degradable linkagesin the polymer backbone or in the linker group between the polymerbackbone and one or more of the terminal functional groups of thepolymer molecule. Such degradable linkages include, but are not limitedto, ester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on a biologicallyactive agent, wherein such ester groups generally hydrolyze underphysiological conditions to release the biologically active agent. Otherhydrolytically degradable linkages include but are not limited tocarbonate linkages; imine linkages resulted from reaction of an amineand an aldehyde; phosphate ester linkages formed by reacting an alcoholwith a phosphate group; hydrazone linkages which are reaction product ofa hydrazide and an aldehyde; acetal linkages that are the reactionproduct of an aldehyde and an alcohol; orthoester linkages that are thereaction product of a formate and an alcohol; peptide linkages formed byan amine group, including but not limited to, at an end of a polymersuch as PEG, and a carboxyl group of a peptide; and oligonucleotidelinkages formed by a phosphoramidite group, including but not limitedto, at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide.

The terms “medium” or “media,” as used herein, refer to any culturemedium used to grow and harvest cells and/or products expressed and/orsecreted by such cells. Such “medium” or “media” include, but are notlimited to, solution, solid, semi-solid, or rigid supports that maysupport or contain any host cell, including, by way of example,bacterial host cells, yeast host cells, insect host cells, plant hostcells, eukaryotic host cells, mammalian host cells, CHO cells,prokaryotic host cells, E. coli, or Pseudomonas host cells, and cellcontents. Such “medium” or “media” includes, but is not limited to,medium or media in which the host cell has been grown into which apolypeptide has been secreted, including medium either before or after aproliferation step. Such “medium” or “media” also includes, but is notlimited to, buffers or reagents that contain host cell lysates, by wayof example a polypeptide produced intracellularly and the host cells arelysed or disrupted to release the polypeptide.

The term “metabolite,” as used herein, refers to a derivative of acompound, by way of example natural amino acid polypeptide, anon-natural amino acid polypeptide, a modified natural amino acidpolypeptide, or a modified non-natural amino acid polypeptide, that isformed when the compound, by way of example natural amino acidpolypeptide, non-natural amino acid polypeptide, modified natural aminoacid polypeptide, or modified non-natural amino acid polypeptide, ismetabolized. The term “pharmaceutically active metabolite” or “activemetabolite” refers to a biologically active derivative of a compound, byway of example natural amino acid polypeptide, a non-natural amino acidpolypeptide, a modified natural amino acid polypeptide, or a modifiednon-natural amino acid polypeptide, that is formed when such a compound,by way of example a natural amino acid polypeptide, non-natural aminoacid polypeptide, modified natural amino acid polypeptide, or modifiednon-natural amino acid polypeptide, is metabolized.

The term “metabolized,” as used herein, refers to the sum of theprocesses by which a particular substance is changed by an organism.Such processes include, but are not limited to, hydrolysis reactions andreactions catalyzed by enzymes. Further information on metabolism may beobtained from The Pharmacological Basis of Therapeutics, 9th Edition,McGraw-Hill (1996). By way of example only, metabolites of natural aminoacid polypeptides, non-natural amino acid polypeptides, modified naturalamino acid polypeptides, or modified non-natural amino acid polypeptidesmay be identified either by administration of the natural amino acidpolypeptides, non-natural amino acid polypeptides, modified naturalamino acid polypeptides, or modified non-natural amino acid polypeptidesto a host and analysis of tissue samples from the host, or by incubationof natural amino acid polypeptides, non-natural amino acid polypeptides,modified natural amino acid polypeptides, or modified non-natural aminoacid polypeptides with hepatic cells in vitro and analysis of theresulting compounds.

The term “metal chelator,” as used herein, refers to a molecule whichforms a metal complex with metal ions. By way of example, such moleculesmay form two or more coordination bonds with a central metal ion and mayform ring structures.

The term “metal-containing moiety,” as used herein, refers to a groupwhich contains a metal ion, atom or particle. Such moieties include, butare not limited to, cisplatin, chelated metals ions (such as nickel,iron, and platinum), and metal nanoparticles (such as nickel, iron, andplatinum).

The term “moiety incorporating a heavy atom,” as used herein, refers toa group which incorporates an ion of atom which is usually heavier thancarbon. Such ions or atoms include, but are not limited to, silicon,tungsten, gold, lead, and uranium.

The term “modified,” as used herein refers to the presence of a changeto a natural amino acid, a non-natural amino acid, a natural amino acidpolypeptide or a non-natural amino acid polypeptide. Such changes, ormodifications, may be obtained by post synthesis modifications ofnatural amino acids, non-natural amino acids, natural amino acidpolypeptides or non-natural amino acid polypeptides, or byco-translational, or by post-translational modification of natural aminoacids, non-natural amino acids, natural amino acid polypeptides ornon-natural amino acid polypeptides. The form “modified or unmodified”means that the natural amino acid, non-natural amino acid, natural aminoacid polypeptide or non-natural amino acid polypeptide being discussedare optionally modified, that is, he natural amino acid, non-naturalamino acid, natural amino acid polypeptide or non-natural amino acidpolypeptide under discussion can be modified or unmodified.

As used herein, the term “modulated serum half-life” refers to positiveor negative changes in the circulating half-life of a modifiedbiologically active molecule relative to its non-modified form. By wayof example, the modified biologically active molecules include, but arenot limited to, natural amino acid, non-natural amino acid, naturalamino acid polypeptide or non-natural amino acid polypeptide. By way ofexample, serum half-life is measured by taking blood samples at varioustime points after administration of the biologically active molecule ormodified biologically active molecule, and determining the concentrationof that molecule in each sample. Correlation of the serum concentrationwith time allows calculation of the serum half-life. By way of example,modulated serum half-life may be an increased in serum half-life, whichmay enable an improved dosing regimens or avoid toxic effects. Suchincreases in serum may be at least about two fold, at least aboutthree-fold, at least about five-fold, or at least about ten-fold. Anon-limiting example of a method to evaluate increases in serumhalf-life is given in examples 88-92. This method may be used forevaluating the serum half-life of any polypeptide.

The term “modulated therapeutic half-life,” as used herein, refers topositive or negative change in the half-life of the therapeuticallyeffective amount of a modified biologically active molecule, relative toits non-modified form. By way of example, the modified biologicallyactive molecules include, but are not limited to, natural amino acid,non-natural amino acid, natural amino acid polypeptide or non-naturalamino acid polypeptide. By way of example, therapeutic half-life ismeasured by measuring pharmacokinetic and/or pharmacodynamic propertiesof the molecule at various time points after administration. Increasedtherapeutic half-life may enable a particular beneficial dosing regimen,a particular beneficial total dose, or avoids an undesired effect. Byway of example, the increased therapeutic half-life may result fromincreased potency, increased or decreased binding of the modifiedmolecule to its target, an increase or decrease in another parameter ormechanism of action of the non-modified molecule, or an increased ordecreased breakdown of the molecules by enzymes such as, by way ofexample only, proteases. A non-limiting example of a method to evaluateincreases in therapeutic half-life is given in examples 88-92. Thismethod may be used for evaluating the therapeutic half-life of anypolypeptide.

The term “nanoparticle,” as used herein, refers to a particle which hasa particle size between about 500 nm to about 1 nm.

The term “near-stoichiometric,” as used herein, refers to the ratio ofthe moles of compounds participating in a chemical reaction being about0.75 to about 1.5.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. By way of example, a non-eukaryotic organism may belong tothe Eubacteria, (which includes but is not limited to, Escherichia coli,Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), phylogeneticdomain, or the Archaea, which includes, but is not limited to,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii,Aeuropyrum pemix, or Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, or phylogenetic domain.

A “non-natural amino acid” refers to an amino acid that is not one ofthe 20 common amino acids or pyrolysine or selenocysteine. Other termsthat may be used synonymously with the term “non-natural amino acid” is“non-naturally encoded amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-natural amino acid”includes, but is not limited to, amino acids which occur naturally bymodification of a naturally encoded amino acid (including but notlimited to, the common amino acids or pyrrolysine and selenocysteine)but are not themselves incorporated into a growing polypeptide chain bythe translation complex. Examples of naturally-occurring amino acidsthat are not naturally-encoded include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine. Additionally, the term “non-natural amino acid”includes, but is not limited to, amino acids which do not occurnaturally and may be obtained synthetically or may be obtained bymodification of non-natural amino acids.

The term “nucleic acid,” as used herein, refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides or ribonucleotides and polymersthereof in either single- or double-stranded form. By way of exampleonly, such nucleic acids and nucleic acid polymers include, but are notlimited to, (i) analogues of natural nucleotides which have similarbinding properties as a reference nucleic acid and are metabolized in amanner similar to naturally occurring nucleotides; (ii) oligonucleotideanalogs including, but are not limited to, PNA (peptidonucleic acid),analogs of DNA used in antisense technology (phosphorothioates,phosphoroamidates, and the like); (iii) conservatively modified variantsthereof (including but not limited to, degenerate codon substitutions)and complementary sequences and sequence explicitly indicated. By way ofexample, degenerate codon substitutions may be achieved by generatingsequences in which the third position of one or more selected (or all)codons is substituted with mixed-base and/or deoxyinosine residues(Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J.Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.Probes 8:91-98 (1994)).

The term “oxidizing agent,” as used herein, refers to a compound ormaterial which is capable of removing an electron from a compound beingoxidized. By way of example oxidizing agents include, but are notlimited to, oxidized glutathione, cystine, cystamine, oxidizeddithiothreitol, oxidized erythreitol, and oxygen. A wide variety ofoxidizing agents are suitable for use in the methods and compositionsdescribed herein.

The term “pharmaceutically acceptable”, as used herein, refers to amaterial, including but not limited, to a salt, carrier or diluent,which does not abrogate the biological activity or properties of thecompound, and is relatively nontoxic, i.e., the material may beadministered to an individual without causing undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the composition in which it is contained.

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity. By way of example only, such alinkage may be covalent or non-covalent.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photocleavable group,” as used herein, refers to a group whichbreaks upon exposure to light.

The term “photocrosslinker,” as used herein, refers to a compoundcomprising two or more functional groups which, upon exposure to light,are reactive and form a covalent or non-covalent linkage with two ormore monomeric or polymeric molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “polyalkylene glycol,” as used herein, refers to linear orbranched polymeric polyether polyols. Such polyalkylene glycols,including, but are not limited to, polyethylene glycol, polypropyleneglycol, polybutylene glycol, and derivatives thereof. Other exemplaryembodiments are listed, for example, in commercial supplier catalogs,such as Shearwater Corporation's catalog “Polyethylene Glycol andDerivatives for Biomedical Applications” (2001). By way of example only,such polymeric polyether polyols have average molecular weights betweenabout 0.1 kDa to about 100 kDa. By way of example, such polymericpolyether polyols include, but are not limited to, between about 100 Daand about 100,000 Da or more. The molecular weight of the polymer may bebetween about 100 Da and about 100,000 Da, including but not limited to,100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da,70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. The molecular weight of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and 50,000Da. In some embodiments, the molecular weight of the branched chain PEGis between about 1,000 Da and 40,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 5,000 Da and40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and 20,000 Da.

The term “polymer,” as used herein, refers to a molecule composed ofrepeated subunits. Such molecules include, but are not limited to,polypeptides, polynucleotides, or polysaccharides or polyalkyleneglycols.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-natural amino acid. Additionally, such “polypeptides,” “peptides”and “proteins” include amino acid chains of any length, including fulllength proteins, wherein the amino acid residues are linked by covalentpeptide bonds.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid which occurs after such an amino acidhas been translationally incorporated into a polypeptide chain. Suchmodifications include, but are not limited to, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

The terms “prodrug” or “pharmaceutically acceptable prodrug,” as usedherein, refers to an agent that is converted into the parent drug invivo or in vitro, wherein which does not abrogate the biologicalactivity or properties of the drug, and is relatively nontoxic, i.e.,the material may be administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.Prodrugs are generally drug precursors that, following administration toa subject and subsequent absorption, are converted to an active, or amore active species via some process, such as conversion by a metabolicpathway. Some prodrugs have a chemical group present on the prodrug thatrenders it less active and/or confers solubility or some other propertyto the drug. Once the chemical group has been cleaved and/or modifiedfrom the prodrug the active drug is generated. Prodrugs are convertedinto active drug within the body through enzymatic or non-enzymaticreactions. Prodrugs may provide improved physiochemical properties suchas better solubility, enhanced delivery characteristics, such asspecifically targeting a particular cell, tissue, organ or ligand, andimproved therapeutic value of the drug. The benefits of such prodrugsinclude, but are not limited to, (i) ease of administration comparedwith the parent drug; (ii) the prodrug may be bioavailable by oraladministration whereas the parent is not; and (iii) the prodrug may alsohave improved solubility in pharmaceutical compositions compared withthe parent drug. A pro-drug includes a pharmacologically inactive, orreduced-activity, derivative of an active drug. Prodrugs may be designedto modulate the amount of a drug or biologically active molecule thatreaches a desired site of action through the manipulation of theproperties of a drug, such as physiochemical, biopharmaceutical, orpharmacokinetic properties. An example, without limitation, of a prodrugwould be a non-natural amino acid polypeptide which is administered asan ester (the “prodrug”) to facilitate transmittal across a cellmembrane where water solubility is detrimental to mobility but whichthen is metabolically hydrolyzed to the carboxylic acid, the activeentity, once inside the cell where water-solubility is beneficial.Prodrugs may be designed as reversible drug derivatives, for use asmodifiers to enhance drug transport to site-specific tissues.

The term “prophylactically effective amount,” as used herein, refersthat amount of a composition containing at least one non-natural aminoacid polypeptide or at least one modified non-natural amino acidpolypeptide prophylactically applied to a patient which will relieve tosome extent one or more of the symptoms of a disease, condition ordisorder being treated. In such prophylactic applications, such amountsmay depend on the patient's state of health, weight, and the like. It isconsidered well within the skill of the art for one to determine suchprophylactically effective amounts by routine experimentation,including, but not limited to, a dose escalation clinical trial.

The term “protected,” as used herein, refers to the presence of a“protecting group” or moiety that prevents reaction of the chemicallyreactive functional group under certain reaction conditions. Theprotecting group will vary depending on the type of chemically reactivegroup being protected. By way of example only, (i) if the chemicallyreactive group is an amine or a hydrazide, the protecting group may beselected from tert-butyloxycarbonyl (t-Boc) and9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive groupis a thio, the protecting group may be orthopyridyldisulfide; and (iii)if the chemically reactive group is a carboxylic acid, such as butanoicor propionic acid, or a hydroxyl group, the protecting group may bebenzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

By way of example only, blocking/protecting groups may also be selectedfrom:

Additionally, protecting groups include, but are not limited to,including photolabile groups such as Nvoc and MeNvoc and otherprotecting groups known in the art. Other protecting groups aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporatedherein by reference in its entirety.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

The term “reactive compound,” as used herein, refers to a compound whichunder appropriate conditions is reactive toward another atom, moleculeor compound.

The term “recombinant host cell,” also referred to as “host cell,”refers to a cell which includes an exogenous polynucleotide, wherein themethods used to insert the exogenous polynucleotide into a cell include,but are not limited to, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. By way ofexample only, such exogenous polynucleotide may be a nonintegratedvector, including but not limited to a plasmid, or may be integratedinto the host genome.

The term “redox-active agent,” as used herein, refers to a moleculewhich oxidizes or reduces another molecule, whereby the redox activeagent becomes reduced or oxidized. Examples of redox active agentinclude, but are not limited to, ferrocene, quinones, Ru2+/3+ complexes,Co2+/3+ complexes, and Os2+/3+ complexes.

The term “reducing agent,” as used herein, refers to a compound ormaterial which is capable of adding an electron to a compound beingreduced. By way of example reducing agents include, but are not limitedto, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine,cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducingagents may be used, by way of example only, to maintain sulfhydrylgroups in the reduced state and to reduce intra- or intermoleculardisulfide bonds.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms an improperly folded or unfolded state to a native orproperly folded conformation. By way of example only, refoldingtransforms disulfide bond containing polypeptides from an improperlyfolded or unfolded state to a native or properly folded conformationwith respect to disulfide bonds. Such disulfide bond containingpolypeptides may be natural amino acid polypeptides or non-natural aminoacid polypeptides.

The term “resin,” as used herein, refers to high molecular weight,insoluble polymer beads. By way of example only, such beads may be usedas supports for solid phase peptide synthesis, or sites for attachmentof molecules prior to purification.

The term “saccharide,” as used herein, refers to a series ofcarbohydrates including but not limited to sugars, monosaccharides,oligosaccharides, and polysaccharides.

The term “safety” or “safety profile,” as used herein, refers to sideeffects that might be related to administration of a drug relative tothe number of times the drug has been administered. By way of example, adrug which has been administered many times and produced only mild or noside effects is said to have an excellent safety profile. A non-limitingexample of a method to evaluate the safety profile is given in example92. This method may be used for evaluating the safety profile of anypolypeptide.

The phrase “selectively hybridizes to” or “specifically hybridizes to,”as used herein, refers to the binding, duplexing, or hybridizing of amolecule to a particular nucleotide sequence under stringenthybridization conditions when that sequence is present in a complexmixture including but not limited to, total cellular or library DNA orRNA.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that can be detected by electron spinresonance spectroscopy and can be attached to another molecule. Suchspin-label molecules include, but are not limited to, nitryl radicalsand nitroxides, and may be single spin-labels or double spin-labels.

The term “stoichiometric,” as used herein, refers to the ratio of themoles of compounds participating in a chemical reaction being about 0.9to about 1.1.

The term “stoichiometric-like,” as used herein, refers to a chemicalreaction which becomes stoichiometric or near-stoichiometric uponchanges in reaction conditions or in the presence of additives. Suchchanges in reaction conditions include, but are not limited to, anincrease in temperature or change in pH. Such additives include, but arenot limited to, accelerants.

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA or other nucleic acid mimics, orcombinations thereof, under conditions of low ionic strength and hightemperature. By way of example, under stringent conditions a probe willhybridize to its target subsequence in a complex mixture of nucleic acid(including but not limited to, total cellular or library DNA or RNA) butdoes not hybridize to other sequences in the complex mixture. Stringentconditions are sequence-dependent and will be different in differentcircumstances. By way of example, longer sequences hybridizespecifically at higher temperatures. Stringent hybridization conditionsinclude, but are not limited to, (i) about 5-10o C lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH; (ii) the salt concentration is about 0.01 M to about1.0 M at about pH 7.0 to about pH 8.3 and the temperature is at leastabout 30oC for short probes (including but not limited to, 10 to 50nucleotides) and at least about 60o C for long probes (including but notlimited to, greater than 50 nucleotides); (iii) the addition ofdestabilizing agents including, but not limited to, formamide, (iv) 50%formamide, 5×SSC, and 1% SDS, incubating at 42oC, or 5×SSC, 1% SDS,incubating at 65oC, with wash in 0.2×SSC, and 0.1% SDS at 65oC forbetween about 5 minutes to about 120 minutes. By way of example only,detection of selective or specific hybridization, includes, but is notlimited to, a positive signal at least two times background. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).

The term “subject” as used herein, refers to an animal which is theobject of treatment, observation or experiment. By way of example only,a subject may be, but is not limited to, a mammal including, but notlimited to, a human.

The term “substantially purified,” as used herein, refers to a componentof interest that may be substantially or essentially free of othercomponents which normally accompany or interact with the component ofinterest prior to purification. By way of example only, a component ofinterest may be “substantially purified” when the preparation of thecomponent of interest contains less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating components. Thus, a“substantially purified” component of interest may have a purity levelof about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98%, about 99% or greater. By way of exampleonly, a natural amino acid polypeptide or a non-natural amino acidpolypeptide may be purified from a native cell, or host cell in the caseof recombinantly produced natural amino acid polypeptides or non-naturalamino acid polypeptides. By way of example a preparation of a naturalamino acid polypeptide or a non-natural amino acid polypeptide may be“substantially purified” when the preparation contains less than about30%, less than about 25%, less than about 20%, less than about 15%, lessthan about 10%, less than about 5%, less than about 4%, less than about3%, less than about 2%, or less than about 1% (by dry weight) ofcontaminating material. By way of example when a natural amino acidpolypeptide or a non-natural amino acid polypeptide is recombinantlyproduced by host cells, the natural amino acid polypeptide ornon-natural amino acid polypeptide may be present at about 30%, about25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%,about 2%, or about 1% or less of the dry weight of the cells. By way ofexample when a natural amino acid polypeptide or a non-natural aminoacid polypeptide is recombinantly produced by host cells, the naturalamino acid polypeptide or non-natural amino acid polypeptide may bepresent in the culture medium at about 5 g/L, about 4 g/L, about 3 g/L,about 2 g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L orless of the dry weight of the cells. By way of example, “substantiallypurified” natural amino acid polypeptides or non-natural amino acidpolypeptides may have a purity level of about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater asdetermined by appropriate methods, including, but not limited to,SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

The term “substituents” also referred to as “non-interferingsubstituents” “refers to groups which may be used to replace anothergroup on a molecule. Such groups include, but are not limited to, halo,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂aralkyl, C₃-C₁₂ cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substitutedphenyl, toluolyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂alkoxyaryl, C₅-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl,C₁-C₁₀ alkylsulfonyl, —(CH₂)m-O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₆-C₁₀ aryl), —(CH₂)m-O—(CH₂)m-O—(C₁-C₁₀ alkyl) whereineach m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂,—NRC(S)NR₂, salts thereof, and the like. Each R group in the precedinglist includes, but is not limited to, H, alkyl or substituted alkyl,aryl or substituted aryl, or alkaryl. Where substituent groups arespecified by their conventional chemical formulas, written from left toright, they equally encompass the chemically identical substituents thatwould result from writing the structure from right to left, for example,—CH₂O— is equivalent to —OCH₂—.

By way of example only, substituents for alkyl and heteroalkyl radicals(including those groups referred to as alkylene, alkenyl,heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl,cycloalkenyl, and heterocycloalkenyl) includes, but is not limited to:—OR, ═O, ═NR, ═N—OR, —NR₂, —SR, -halogen, —SiR₃, —OC(O)R, —C(O)R, —CO₂R,—CONR₂, —OC(O)NR₂, —NRC(O)R, —NRC(O)NR₂, —NR(O)₂R, —NR—C(NR₂)═NR,—S(O)R, —S(O)₂R, —S(O)₂NR₂, —NRSO₂R, —CN and —NO₂. Each R group in thepreceding list includes, but is not limited to, hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, includingbut not limited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups.When two R groups are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR2 is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl.

By way of example, substituents for aryl and heteroaryl groups include,but are not limited to, —OR, ═O, ═NR, ═N—OR, —NR₂, —SR, -halogen, —SiR₃,—OC(O)R, —C(O)R, —CO₂R, —CONR₂, —OC(O)NR₂, —NRC(O)R, —NRC(O)NR₂,—NR(O)₂R, —NR—C(NR₂)═NR, —S(O)R, —S(O)₂R, —S(O)₂NR₂, —NRSO₂R, —CN, —NO₂,—R, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in anumber ranging from zero to the total number of open valences on thearomatic ring system; and where each R group in the preceding listincludes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl andheteroaryl.

The term “therapeutically effective amount,” as used herein, refers tothe amount of a composition containing at least one non-natural aminoacid polypeptide and/or at least one modified non-natural amino acidpolypeptide administered to a patient already suffering from a disease,condition or disorder, sufficient to cure or at least partially arrest,or relieve to some extent one or more of the symptoms of the disease,disorder or condition being treated. The effectiveness of suchcompositions depend conditions including, but not limited to, theseverity and course of the disease, disorder or condition, previoustherapy, the patient's health status and response to the drugs, and thejudgment of the treating physician. By way of example only,therapeutically effective amounts may be determined by routineexperimentation, including but not limited to a dose escalation clinicaltrial.

The term “thioalkoxy,” as used herein, refers to sulfur containing alkylgroups linked to molecules via an oxygen atom.

The term “thermal melting point” or Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of probescomplementary to a target hybridize to the target sequence atequilibrium.

The term “toxic moiety,” as used herein, refers to a compound which cancause harm or death.

The terms “treat,” “treating” or “treatment”, as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition. The terms “treat,”“treating” or “treatment”, include, but are not limited to, prophylacticand/or therapeutic treatments.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Such water soluble polymersinclude, but are not limited to, polyethylene glycol, polyethyleneglycol propionaldehyde, mono C₁-C₁₀ alkoxy or aryloxy derivativesthereof (described in U.S. Pat. No. 5,252,714 which is incorporated byreference herein), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleicanhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextranderivatives including dextran sulfate, polypropylene glycol,polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol,heparin, heparin fragments, polysaccharides, oligosaccharides, glycans,cellulose and cellulose derivatives, including but not limited tomethylcellulose and carboxymethyl cellulose, serum albumin, starch andstarch derivatives, polypeptides, polyalkylene glycol and derivativesthereof, copolymers of polyalkylene glycols and derivatives thereof,polyvinyl ethyl ethers, andalpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. By way of example only, coupling of such water solublepolymers to natural amino acid polypeptides or non-natural polypeptidesmay result in changes including, but not limited to, increased watersolubility, increased or modulated serum half-life, increased ormodulated therapeutic half-life relative to the unmodified form,increased bioavailability, modulated biological activity, extendedcirculation time, modulated immunogenicity, modulated physicalassociation characteristics including, but not limited to, aggregationand multimer formation, altered receptor binding, altered binding to oneor more binding partners, and altered receptor dimerization ormultimerization. In addition, such water soluble polymers may or may nothave their own biological activity.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

Compounds, (including, but not limited to non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides, and reagents for producing the aforementioned compounds)presented herein include isotopically-labeled compounds, which areidentical to those recited in the various formulas and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labeled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

Some of the compounds herein (including, but not limited to non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides, and reagents for producing theaforementioned compounds) have asymmetric carbon atoms and can thereforeexist as enantiomers or diastereomers. Diasteromeric mixtures can beseparated into their individual diastereomers on the basis of theirphysical chemical differences by methods known, for example, bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diastereomers and converting (e.g.,hydrolyzing) the individual diastereomers to the corresponding pureenantiomers. All such isomers, including diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein.

In additional or further embodiments, the compounds described herein(including, but not limited to non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acidpolypeptides, and reagents for producing the aforementioned compounds)are used in the form of pro-drugs. In additional or further embodiments,the compounds described herein ((including, but not limited tonon-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides, and reagents for producingthe aforementioned compounds) are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-natural amino acidsand “modified or unmodified” non-natural amino acid polypeptides.

The methods and formulations described herein include the use ofN-oxides, crystalline forms (also known as polymorphs), orpharmaceutically acceptable salts of non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides. In certain embodiments, non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides may exist as tautomers. All tautomers are included withinthe scope of the non-natural amino acids, non-natural amino acidpolypeptides and modified non-natural amino acid polypeptides presentedherein. In addition, the non-natural amino acids, non-natural amino acidpolypeptides and modified non-natural amino acid polypeptides describedherein can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. The solvated forms of the non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acid polypeptidespresented herein are also considered to be disclosed herein.

Some of the compounds herein (including, but not limited to non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides and reagents for producing theaforementioned compounds) may exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein. Also, for example all enol-keto forms of any compounds(including, but not limited to non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acid polypeptidesand reagents for producing the aforementioned compounds) herein areconsidered as part of the compositions described herein.

Some of the compounds herein (including, but not limited to non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides and reagents for producing either ofthe aforementioned compounds) are acidic and may form a salt with apharmaceutically acceptable cation. Some of the compounds herein(including, but not limited to non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acid polypeptidesand reagents for producing the aforementioned compounds) can be basicand accordingly, may form a salt with a pharmaceutically acceptableanion. All such salts, including di-salts are within the scope of thecompositions described herein and they can be prepared by conventionalmethods. For example, salts can be prepared by contacting the acidic andbasic entities, in either an aqueous, non-aqueous or partially aqueousmedium. The salts are recovered by using at least one of the followingtechniques: filtration, precipitation with a non-solvent followed byfiltration, evaporation of the solvent, or, in the case of aqueoussolutions, lyophilization.

Pharmaceutically acceptable salts of the non-natural amino acidpolypeptides disclosed herein may be formed when an acidic protonpresent in the parent non-natural amino acid polypeptides either isreplaced by a metal ion, by way of example an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase. In addition, the salt forms of the disclosed non-natural aminoacid polypeptides can be prepared using salts of the starting materialsor intermediates. The non-natural amino acid polypeptides describedherein may be prepared as a pharmaceutically acceptable acid additionsalt (which is a type of a pharmaceutically acceptable salt) by reactingthe free base form of non-natural amino acid polypeptides describedherein with a pharmaceutically acceptable inorganic or organic acid.Alternatively, the non-natural amino acid polypeptides described hereinmay be prepared as pharmaceutically acceptable base addition salts(which is a type of a pharmaceutically acceptable salt) by reacting thefree acid form of non-natural amino acid polypeptides described hereinwith a pharmaceutically acceptable inorganic or organic base.

The type of pharmaceutical acceptable salts, include, but are notlimited to: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; (2) salts formed when anacidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. Acceptable inorganicbases include aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like.

The corresponding counterions of the non-natural amino acid polypeptidepharmaceutical acceptable salts may be analyzed and identified usingvarious methods including, but not limited to, ion exchangechromatography, ion chromatography, capillary electrophoresis,inductively coupled plasma, atomic absorption spectroscopy, massspectrometry, or any combination thereof. In addition, the therapeuticactivity of such non-natural amino acid polypeptide pharmaceuticalacceptable salts may be tested using the techniques and methodsdescribed in examples 87-91.

It should be understood that a reference to a salt includes the solventaddition forms or crystal forms thereof, particularly solvates orpolymorphs. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and are often formed during the process ofcrystallization with pharmaceutically acceptable solvents such as water,ethanol, and the like. Hydrates are formed when the solvent is water, oralcoholates are formed when the solvent is alcohol. Polymorphs includethe different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate.

The screening and characterization of non-natural amino acid polypeptidepharmaceutical acceptable salts polymorphs and/or solvates may beaccomplished using a variety of techniques including, but not limitedto, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption,and microscopy. Thermal analysis methods address thermo chemicaldegradation or thermo physical processes including, but not limited to,polymorphic transitions, and such methods are used to analyze therelationships between polymorphic forms, determine weight loss, to findthe glass transition temperature, or for excipient compatibilitystudies. Such methods include, but are not limited to, Differentialscanning calorimetry (DSC), Modulated Differential Scanning Calorimetry(MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric andInfrared analysis (TG/IR). X-ray diffraction methods include, but arenot limited to, single crystal and powder diffractometers andsynchrotron sources. The various spectroscopic techniques used include,but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solidstate). The various microscopy techniques include, but are not limitedto, polarized light microscopy, Scanning Electron Microscopy (SEM) withEnergy Dispersive X-Ray Analysis (EDX), Environmental Scanning ElectronMicroscopy with EDX (in gas or water vapor atmosphere), IR microscopy,and Raman microscopy.

BRIEF DESCRIPTION OF THE FIGURES

A better understanding of the features and advantages of the presentmethods and compositions may be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of our methods, compositions, devices and apparatuses areutilized, and the accompanying drawings of which:

FIG. 1 presents a schematic representation of the relationship ofcertain aspects of the methods, compositions, strategies and techniquesdescribed herein.

FIG. 2 presents illustrative, non-limiting examples of the types ofnon-natural amino acids described herein. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 3 presents illustrative, non-limiting examples of the types ofnon-natural amino acids described herein. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 4 presents an illustrative, non-limiting example of the syntheticmethodology used to make the non-natural amino acids described herein.Such non-natural amino acids may be used in or incorporated into any ofthe methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 5 presents illustrative, non-limiting examples of the syntheticmethodology used to make the non-natural amino acids described herein.Such non-natural amino acids may be used in or incorporated into any ofthe methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 6 presents illustrative, non-limiting examples of the syntheticmethodology used to make the non-natural amino acids described herein.Such non-natural amino acids may be used in or incorporated into any ofthe methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 7 presents illustrative, non-limiting examples of thepost-translational modification of carbonyl-containing non-natural aminoacid polypeptides with hydroxylamine-containing reagents to formmodified oxime-containing non-natural amino acid polypeptides. Suchnon-natural amino acid polypeptides may be used in or incorporated intoany of the methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 8 presents illustrative, non-limiting examples of additives thatcan be used to enhance the reaction of carbonyl-containing non-naturalamino acid polypeptides with hydroxylamine-containing reagents to formmodified oxime-containing non-natural amino acid polypeptides.

FIG. 9 presents illustrative, non-limiting examples of thepost-translational modification of oxime-containing non-natural aminoacid polypeptides with carbonyl-containing reagents to form modifiedoxime-containing non-natural amino acid polypeptides. Such non-naturalamino acid polypeptides may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 10 presents an illustrative, non-limiting example of thepost-translational modification of hydroxylamine-containing non-naturalamino acid polypeptides with carbonyl-containing reagents to formmodified oxime-containing non-natural amino acid polypeptides. Suchnon-natural amino acid polypeptides may be used in or incorporated intoany of the methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 11 presents illustrative, non-limiting examples of PEG-containingreagents that can be used to modify non-natural amino acid polypeptidesto form PEG-containing, oxime-linked non-natural amino acidpolypeptides.

FIG. 12 presents illustrative, non-limiting examples of the synthesis ofPEG-containing reagents that can be used to modify non-natural aminoacid polypeptides to form PEG-containing, oxime-linked non-natural aminoacid polypeptides.

FIG. 13 presents an illustrative, non-limiting example of the synthesisof an amide-based PEG-containing reagent that can be used to modifynon-natural amino acid polypeptides to form PEG-containing, oxime-linkednon-natural amino acid polypeptides.

FIG. 14 presents an illustrative, non-limiting example of the synthesisof a carbamate-based PEG-containing reagent that can be used to modifynon-natural amino acid polypeptides to form PEG-containing, oxime-linkednon-natural amino acid polypeptides.

FIG. 15 presents an illustrative, non-limiting example of the synthesisof a carbamate-based PEG-containing reagent that can be used to modifynon-natural amino acid polypeptides to form PEG-containing, oxime-linkednon-natural amino acid polypeptides.

FIG. 16 presents illustrative, non-limiting examples of the synthesis ofsimple PEG-containing reagents that can be used to modify non-naturalamino acid polypeptides to form PEG-containing, oxime-linked non-naturalamino acid polypeptides.

FIG. 17 presents illustrative, non-limiting examples of branchedPEG-containing reagents that can be used to modify non-natural aminoacid polypeptides to form PEG-containing, oxime-linked non-natural aminoacid polypeptides, and the use of one such reagent to modify acarbonyl-based non-natural amino acid polypeptide.

FIG. 18 presents an illustrative, non-limiting example of the synthesisof a bifunctional linker group that can be used to modify and linknon-natural amino acid polypeptides.

FIG. 19 presents illustrative, non-limiting examples of multifunctionallinker groups that can be used to modify and link non-natural amino acidpolypeptides.

FIG. 20 presents an illustrative, non-limiting representation of the useof a bifunctional linker group to modify and link a non-natural aminoacid polypeptide to a PEG group.

FIG. 21 presents illustrative, non-limiting examples of the use ofbifunctional linker groups to modify and link non-natural amino acidpolypeptides to a PEG group.

FIG. 22 presents an illustrative, non-limiting representation of the useof a bifunctional linker group to link together two non-natural aminoacid polypeptides to form a homodimer.

FIG. 23 presents an illustrative, non-limiting representation of the useof a bifunctional linker group to link together two differentnon-natural amino acid polypeptides to form a heterodimer.

FIG. 24 presents an illustrative, non-limiting representation of thesynthesis of a carbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 25 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 26 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid.

FIG. 27 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 28 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 29 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 30 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 31 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 32 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 33 presents an illustrative, non-limiting representation of thesynthesis of a dicarbonyl-containing non-natural amino acid. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 34 presents illustrative, non-limiting representations of thesyntheses of dicarbonyl-containing non-natural amino acids. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 35 presents illustrative, non-limiting representations of carbonyl-and dicarbonyl-containing non-natural amino acids. Such non-naturalamino acids may be used in or incorporated into any of the methods,compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 36 presents illustrative, non-limiting representations of thesyntheses of non-natural amino acids. Such non-natural amino acids maybe used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 37 presents illustrative, non-limiting representations of thesyntheses of carbonyl- and dicarbonyl-containing non-natural aminoacids. Such non-natural amino acids may be used in or incorporated intoany of the methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 38 presents illustrative, non-limiting representations of thesyntheses of carbonyl- and dicarbonyl-containing non-natural aminoacids. Such non-natural amino acids may be used in or incorporated intoany of the methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 39 presents illustrative, non-limiting representations of thesyntheses of carbonyl- and dicarbonyl-containing non-natural aminoacids. Such non-natural amino acids may be used in or incorporated intoany of the methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 40 presents illustrative, non-limiting representations ofdicarbonyl-containing non-natural amino acids. Such non-natural aminoacids may be used in or incorporated into any of the methods,compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 41 presents illustrative, non-limiting representations ofdicarbonyl-containing non-natural amino acids. Such non-natural aminoacids may be used in or incorporated into any of the methods,compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 42 presents illustrative, non-limiting representations ofdicarbonyl-containing non-natural amino acids. Such non-natural aminoacids may be used in or incorporated into any of the methods,compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 43 presents illustrative, non-limiting representations of (a)protected or unprotected 1,3-ketoaldehyde-containing non-natural aminoacids, and (b) 1-3-ketocarboxylyl(thio)ester-containing non-naturalamino acids. Such non-natural amino acids may be used in or incorporatedinto any of the methods, compositions, techniques and strategies formaking, purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 44 presents illustrative, non-limiting representations ofhydrazide-containing non-natural amino acids. Such non-natural aminoacids may be used in or incorporated into any of the methods,compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 45 presents illustrative, non-limiting representations ofhydrazide-containing non-natural amino acids. Such non-natural aminoacids may be used in or incorporated into any of the methods,compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIGS. 46A and 46B presents illustrative, non-limiting representations ofoxime-containing non-natural amino acids, and FIG. 46C presentsillustrative, non-limiting representations of hydrazine-containingnon-natural amino acids. Such non-natural amino acids may be used in orincorporated into any of the methods, compositions, techniques andstrategies for making, purifying, characterizing, and using non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides described herein.

FIG. 47 presents illustrative, non-limiting representations of one-stepconjugation to non-natural amino acid polypeptides and two-stepconjugation to non-natural amino acid polypeptides. By way of example,such conjugations include PEGylation of to non-natural amino acidpolypeptides.

FIG. 48 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 49 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 50 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 51 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 52 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 53 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 54 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 55 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 56 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 57 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 58 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 59 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 60 presents illustrative, non-limiting representations of thesynthesis of hydroxylamine compounds. Such non-natural amino acids maybe used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 61 presents illustrative, non-limiting representations of thesynthesis of mPEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 62A presents illustrative, non-limiting representations of thesynthesis of hydroxylamine compounds; FIG. 62B presents illustrative,non-limiting representations of the synthesis of mPEG compounds. Suchnon-natural amino acids may be used in or incorporated into any of themethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-natural amino acids, non-natural aminoacid polypeptides and modified non-natural amino acid polypeptidesdescribed herein.

FIG. 63 presents illustrative, non-limiting examples of (A) themodification of non-natural amino acid polypeptides by chemicalconversion into carbonyl-containing (including dicarbonyl-containing)non-natural amino acid polypeptides and (B) the modification ofnon-natural amino acid polypeptides by chemical conversion intohydroxylamine-containing non-natural amino acid polypeptides. Suchnon-natural amino acid polypeptides may be used in or incorporated intoany of the methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein.

FIG. 64 presents illustrative, non-limiting representations of thesynthesis of PEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

FIG. 65 presents illustrative, non-limiting representations of thesynthesis of PEG-hydroxylamine compounds. Such non-natural amino acidsmay be used in or incorporated into any of the methods, compositions,techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-natural amino acids to proteins invivo. A number of new amino acids with novel chemical, physical orbiological properties, including photoaffinity labels andphotoisomerizable amino acids, keto amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027 (incorporated by referencein its entirety); J. W. Chin, & P. G. Schultz, (2002), ChemBioChem3(11):1135-1137 (incorporated by reference in its entirety); J. W. Chin,et al., (2002), PNAS United States of America 99(17):11020-11024(incorporated by reference in its entirety); and, L. Wang, & P. G.Schultz, (2002), Chem. Comm. 1-11 (incorporated by reference in itsentirety). These studies have demonstrated that it is possible toselectively and routinely introduce chemical functional groups that arenot found in proteins, that are chemically inert to all of thefunctional groups found in the 20 common, genetically-encoded aminoacids and that may be used to react efficiently and selectively to formstable covalent linkages.

II. Overview

FIG. 1 presents an overview of the compositions, methods and techniquesthat are described herein. At one level, described herein are the tools(methods, compositions, techniques) for creating and using a polypeptidecomprising at least one non-natural amino acid or modified non-naturalamino acid with a carbonyl, dicarbonyl, oxime or hydroxylamine group.Such non-natural amino acids may contain further functionality,including but not limited to, a label; a dye; a polymer; a water-solublepolymer; a derivative of polyethylene glycol; a photocrosslinker; acytotoxic compound; a drug; an affinity label; a photoaffinity label; areactive compound; a resin; a second protein or polypeptide orpolypeptide analog; an antibody or antibody fragment; a metal chelator;a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; aRNA; an antisense polynucleotide; a saccharide, a water-solubledendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label;a fluorophore; a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent (in which case, the biologically activeagent can include an agent with therapeutic activity and the non-naturalamino acid polypeptide or modified non-natural amino acid can serveeither as a co-therapeutic agent with the attached therapeutic agent oras a means for delivery the therapeutic agent to a desired site withinan organism); a detectable label; a small molecule; an inhibitoryribonucleic acid; a radionucleotide; a neutron-capture agent; aderivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof. Note that the various aforementionedfunctionalities are not meant to imply that the members of onefunctionality can not be classified as members of another functionality.Indeed, there will be overlap depending upon the particularcircumstances. By way of example only, a water-soluble polymer overlapsin scope with a derivative of polyethylene glycol, however the overlapis not complete and thus both functionalities are cited above.

As shown in FIG. 1, in one aspect are methods for selecting anddesigning a polypeptide to be modified using the methods, compositionsand techniques described herein. The new polypeptide may be designed denovo, including by way of example only, as part of high-throughputscreening process (in which case numerous polypeptides may be designed,synthesized, characterized and/or tested) or based on the interests ofthe researcher. The new polypeptide may also be designed based on thestructure of a known or partially characterized polypeptide. By way ofexample only, the Growth Hormone Gene Superfamily (see infra) has beenthe subject of intense study by the scientific community; a newpolypeptide may be designed based on the structure of a member ormembers of this gene superfamily. The principles for selecting whichamino acid(s) to substitute and/or modify are described separatelyherein. The choice of which modification to employ is also describedherein, and can be used to meet the need of the experimenter or enduser. Such needs may include, but are not limited to, manipulating thetherapeutic effectiveness of the polypeptide, improving the safetyprofile of the polypeptide, adjusting the pharmacokinetics,pharmacologics and/or pharmacodynamics of the polypeptide, such as, byway of example only, increasing water solubility, bioavailability,increasing serum half-life, increasing therapeutic half-life, modulatingimmunogenicity, modulating biological activity, or extending thecirculation time. In addition, such modifications include, by way ofexample only, providing additional functionality to the polypeptide,incorporating a tag, label or detectable signal into the polypeptide,easing the isolation properties of the polypeptide, and any combinationof the aforementioned modifications.

Also described herein are non-natural amino acids that have or can bemodified to contain an oxime, carbonyl, dicarbonyl, or hydroxylaminegroup. Included with this aspect are methods for producing, purifying,characterizing and using such non-natural amino acids. In another aspectdescribed herein are methods, strategies and techniques forincorporating at least one such non-natural amino acid into apolypeptide. Also included with this aspect are methods for producing,purifying, characterizing and using such polypeptides containing atleast one such non-natural amino acid. Also included with this aspectare compositions of and methods for producing, purifying, characterizingand using oligonucleotides (including DNA and RNA) that can be used toproduce, at least in part, a polypeptide containing at least onenon-natural amino acid. Also included with this aspect are compositionsof and methods for producing, purifying, characterizing and using cellsthat can express such oligonucleotides that can be used to produce, atleast in part, a polypeptide containing at least one non-natural aminoacid.

Thus, polypeptides comprising at least one non-natural amino acid ormodified non-natural amino acid with a carbonyl, dicarbonyl, oxime orhydroxylamine group are provided and described herein. In certainembodiments, polypeptides with at least one non-natural amino acid ormodified non-natural amino acid with a carbonyl, dicarbonyl, oxime orhydroxylamine group include at least one post-translational modificationat some position on the polypeptide. In some embodiments theco-translational or post-translational modification occurs via thecellular machinery (e.g., glycosylation, acetylation, acylation,lipid-modification, palmitoylation, palmitate addition, phosphorylation,glycolipid-linkage modification, and the like), in many instances, suchcellular-machinery-based co-translational or post-translationalmodifications occur at the naturally occurring amino acid sites on thepolypeptide, however, in certain embodiments, thecellular-machinery-based co-translational or post-translationalmodifications occur on the non-natural amino acid site(s) on thepolypeptide.

In other embodiments the post-translational modification does notutilize the cellular machinery, but the functionality is insteadprovided by attachment of a molecule (including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug;an affinity label; a photoaffinity label; a reactive compound; a resin;a second protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin,a biomaterial; a nanoparticle; a spin label; a fluorophore, ametal-containing moiety; a radioactive moiety; a novel functional group;a group that covalently or noncovalently interacts with other molecules;a photocaged moiety; an actinic radiation excitable moiety; a ligand; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof) comprising a second reactive group to the at leastone non-natural amino acid comprising a first reactive group (includingbut not limited to, non-natural amino acid containing a ketone,aldehyde, acetal, hemiacetal, oxime, or hydroxylamine functional group)utilizing chemistry methodology described herein, or others suitable forthe particular reactive groups. In certain embodiments, theco-translational or post-translational modification is made in vivo in aeukaryotic cell or in a non-eukaryotic cell. In certain embodiments, thepost-translational modification is made in vitro not utilizing thecellular machinery. Also included with this aspect are methods forproducing, purifying, characterizing and using such polypeptidescontaining at least one such co-translationally or post-translationallymodified non-natural amino acids.

Also included within the scope of the methods, compositions, strategiesand techniques described herein are reagents capable of reacting with anon-natural amino acid (containing a carbonyl or dicarbonyl group, oximegroup, hydroxylamine group, or masked or protected forms thereof) thatis part of a polypeptide so as to produce any of the aforementionedpost-translational modifications. In general, the resultingpost-translationally modified non-natural amino acid will contain atleast one oxime group; the resulting modified oxime-containingnon-natural amino acid may undergo subsequent modification reactions.Also included with this aspect are methods for producing, purifying,characterizing and using such reagents that are capable of any suchpost-translational modifications of such non-natural amino acid(s).

In certain embodiments, the polypeptide includes at least oneco-translational or post-translational modification that is made in vivoby one host cell, where the post-translational modification is notnormally made by another host cell type. In certain embodiments, thepolypeptide includes at least one co-translational or post-translationalmodification that is made in vivo by a eukaryotic cell, where theco-translational or post-translational modification is not normally madeby a non-eukaryotic cell. Examples of such co-translational orpost-translational modifications include, but are not limited to,glycosylation, acetylation, acylation, lipid-modification,palmitoylation, palmitate addition, phosphorylation, glycolipid-linkagemodification, and the like. In one embodiment, the co-translational orpost-translational modification comprises attachment of anoligosaccharide to an asparagine by a GlcNAc-asparagine linkage(including but not limited to, where the oligosaccharide comprises(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In another embodiment,the co-translational or post-translational modification comprisesattachment of an oligosaccharide (including but not limited to,Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine by aGalNAc-serine, a GalNAc-threonine, a GlcNAc-serine, or aGlcNAc-threonine linkage. In certain embodiments, a protein orpolypeptide can comprise a secretion or localization sequence, anepitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or thelike. Also included with this aspect are methods for producing,purifying, characterizing and using such polypeptides containing atleast one such co-translational or post-translational modification. Inother embodiments, the glycosylated non-natural amino acid polypeptideis produced in a non-glycosylated form. Such a non-glycosylated form ofa glycosylated non-natural amino acid may be produced by methods thatinclude chemical or enzymatic removal of oligosaccharide groups from anisolated or substantially purified or unpurified glycosylatednon-natural amino acid polypeptide; production of the non-natural aminoacid in a host that does not glycosylate such a non-natural amino acidpolypeptide (such a host including, prokaryotes or eukaryotes engineeredor mutated to not glycosylate such a polypeptide), the introduction of aglycosylation inhibitor into the cell culture medium in which such anon-natural amino acid polypeptide is being produced by a eukaryote thatnormally would glycosylate such a polypeptide, or a combination of anysuch methods. Also described herein are such non-glycosylated forms ofnormally-glycosylated non-natural amino acid polypeptides (bynormally-glycosylated is meant a polypeptide that would be glycosylatedwhen produced under conditions in which naturally-occurring polypeptidesare glycosylated). Of course, such non-glycosylated forms ofnormally-glycosylated non-natural amino acid polypeptides (or indeed anypolypeptide described herein) may be in an unpurified form, asubstantially purified form, or in an isolated form.

In certain embodiments, the non-natural amino acid polypeptide includesat least one post-translational modification that is made in thepresence of an accelerant, wherein the post-translational modificationis stoichiometric, stoichiometric-like, or near-stoichiometric. In otherembodiments the polypeptide is contacted with a reagent of Formula (XIX)in the presence of an accelerant. In other embodiments the accelerant isselected from the group consisting of:

The non-natural amino acid containing polypeptide may contain at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, or ten or morenon-natural amino acids containing either a carbonyl or dicarbonylgroup, oxime group, hydroxylamine group, or protected forms thereof. Thenon-natural amino acids can be the same or different, for example, therecan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more different sites in the protein that comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or moredifferent non-natural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with a non-natural aminoacid.

The methods and compositions provided and described herein includepolypeptides comprising at least one non-natural amino acid containing acarbonyl or dicarbonyl group, oxime group, hydroxylamine group, orprotected or masked forms thereof. Introduction of at least onenon-natural amino acid into a polypeptide can allow for the applicationof conjugation chemistries that involve specific chemical reactions,including, but not limited to, with one or more non-natural amino acidswhile not reacting with the commonly occurring 20 amino acids. Onceincorporated, the non-naturally occurring amino acid side chains canalso be modified by utilizing chemistry methodologies described hereinor suitable for the particular functional groups or substituents presentin the naturally encoded amino acid.

The non-natural amino acid methods and compositions described hereinprovide conjugates of substances having a wide variety of functionalgroups, substituents or moieties, with other substances including butnot limited to a label; a dye; a polymer; a water-soluble polymer; aderivative of polyethylene glycol; a photocrosslinker; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide, a water-soluble dendrimer, acyclodextrin, a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof.

In certain embodiments the non-natural amino acids, non-natural aminoacid polypeptides, linkers and reagents described herein, includingcompounds of Formulas (I)-(XXXIII) are stable in aqueous solution undermildly acidic conditions (including but not limited to pH 2 to 8). Inother embodiments, such compounds are stable for at least one monthunder mildly acidic conditions. In other embodiments, such compounds arestable for at least 2 weeks under mildly acidic conditions. In otherembodiments, such compounds are stable for at least 5 days under mildlyacidic conditions.

In another aspect of the compositions, methods, techniques andstrategies described herein are methods for studying or using any of theaforementioned “modified or unmodified” non-natural amino acidpolypeptides. Included within this aspect, by way of example only, aretherapeutic, diagnostic, assay-based, industrial, cosmetic, plantbiology, environmental, energy-production, consumer-products, and/ormilitary uses which would benefit from a polypeptide comprising a“modified or unmodified” non-natural amino acid polypeptide or protein.

III. Location of Non-Natural Amino Acids in Polypeptides

The methods and compositions described herein include incorporation ofone or more non-natural amino acids into a polypeptide. One or morenon-natural amino acids may be incorporated at one or more particularpositions which does not disrupt activity of the polypeptide. This canbe achieved by making “conservative” substitutions, including but notlimited to, substituting hydrophobic amino acids with non-natural ornatural hydrophobic amino acids, bulky amino acids with non-natural ornatural bulky amino acids, hydrophilic amino acids with non-natural ornatural hydrophilic amino acids) and/or inserting the non-natural aminoacid in a location that is not required for activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-natural amino acidwithin the polypeptide. Any position of the polypeptide chain issuitable for selection to incorporate a non-natural amino acid, andselection may be based on rational design or by random selection for anyor no particular desired purpose. Selection of desired sites may bebased on producing a non-natural amino acid polypeptide (which may befurther modified or remain unmodified) having any desired property oractivity, including but not limited to agonists, super-agonists, partialagonists, inverse agonists, antagonists, receptor binding modulators,receptor activity modulators, modulators of binding to binder partners,binding partner activity modulators, binding partner conformationmodulators, dimer or multimer formation, no change to activity orproperty compared to the native molecule, or manipulating any physicalor chemical property of the polypeptide such as solubility, aggregation,or stability. For example, locations in the polypeptide required forbiological activity of a polypeptide can be identified using methodsincluding, but not limited to, point mutation analysis, alanine scanningor homolog scanning methods. Residues other than those identified ascritical to biological activity by methods including, but not limitedto, alanine or homolog scanning mutagenesis may be good candidates forsubstitution with a non-natural amino acid depending on the desiredactivity sought for the polypeptide. Alternatively, the sites identifiedas critical to biological activity may also be good candidates forsubstitution with a non-natural amino acid, again depending on thedesired activity sought for the polypeptide. Another alternative wouldbe to simply make serial substitutions in each position on thepolypeptide chain with a non-natural amino acid and observe the effecton the activities of the polypeptide. Any means, technique, or methodfor selecting a position for substitution with a non-natural amino acidinto any polypeptide is suitable for use in the methods, techniques andcompositions described herein.

The structure and activity of naturally-occurring mutants of apolypeptide that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-natural amino acid. Once residues that are likely to beintolerant to substitution with non-natural amino acids have beeneliminated, the impact of proposed substitutions at each of theremaining positions can be examined using methods including, but notlimited to, the three-dimensional structure of the relevant polypeptide,and any associated ligands or binding proteins. X-ray crystallographicand NMR structures of many polypeptides are available in the ProteinData Bank (PDB, www.rcsb.org), a centralized database containingthree-dimensional structural data of large molecules of proteins andnucleic acids, one can be used to identify amino acid positions that canbe substituted with non-natural amino acids. In addition, models may bemade investigating the secondary and tertiary structure of polypeptides,if three-dimensional structural data is not available. Thus, theidentity of amino acid positions that can be substituted withnon-natural amino acids can be readily obtained.

Exemplary sites of incorporation of a non-natural amino acid include,but are not limited to, those that are excluded from potential receptorbinding regions, or regions for binding to binding proteins or ligandsmay be fully or partially solvent exposed, have minimal or nohydrogen-bonding interactions with nearby residues, may be minimallyexposed to nearby reactive residues, and/or may be in regions that arehighly flexible as predicted by the three-dimensional crystal structureof a particular polypeptide with its associated receptor, ligand orbinding proteins.

A wide variety of non-natural amino acids can be substituted for, orincorporated into, a given position in a polypeptide. By way of example,a particular non-natural amino acid may be selected for incorporationbased on an examination of the three dimensional crystal structure of apolypeptide with its associated ligand, receptor and/or bindingproteins, a preference for conservative substitutions

In one embodiment, the methods described herein include incorporatinginto the polypeptide the non-natural amino acid, where the non-naturalamino acid comprises a first reactive group; and contacting thepolypeptide with a molecule (including but not limited to a label; adye; a polymer; a water-soluble polymer; a derivative of polyethyleneglycol; a photocrosslinker; a cytotoxic compound; a drug; an affinitylabel; a photoaffinity label; a reactive compound; a resin; a secondprotein or polypeptide or polypeptide analog; an antibody or antibodyfragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a ligand; a photoisomerizablemoiety; biotin; a biotin analogue; a moiety incorporating a heavy atom;a chemically cleavable group; a photocleavable group; an elongated sidechain; a carbon-linked sugar; a redox-active agent; an amino thioacid; atoxic moiety; an isotopically labeled moiety; a biophysical probe; aphosphorescent group; a chemiluminescent group; an electron dense group;a magnetic group; an intercalating group; a chromophore; an energytransfer agent; a biologically active agent; a detectable label; a smallmolecule; an inhibitory ribonucleic acid, a radionucleotide; aneutron-capture agent; a derivative of biotin; quantum dot(s); ananotransmitter; a radiotransmitter; an abzyme, an activated complexactivator, a virus, an adjuvant, an aglycan, an allergan, anangiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, asaponin, a shuttle vector, a macromolecule, a mimotope, a receptor, areverse micelle, and any combination thereof) that comprises a secondreactive group. In certain embodiments, the first reactive group is acarbonyl or dicarbonyl moiety and the second reactive group is ahydroxylamine moiety, whereby an oxime linkage is formed. In certainembodiments, the first reactive group is a hydroxylamine moiety and thesecond reactive group is carbonyl or dicarbonyl moiety, whereby an oximelinkage is formed. In certain embodiments, the first reactive group is acarbonyl or dicarbonyl moiety and the second reactive group is an oximemoiety, whereby an oxime exchange reaction occurs. In certainembodiments, the first reactive group is an oxime moiety and the secondreactive group is carbonyl or dicarbonyl moiety, whereby an oximeexchange reaction occurs.

In some cases, the non-natural amino acid substitution(s) orincorporation(s) will be combined with other additions, substitutions,or deletions within the polypeptide to affect other chemical, physical,pharmacologic and/or biological traits. In some cases, the otheradditions, substitutions or deletions may increase the stability(including but not limited to, resistance to proteolytic degradation) ofthe polypeptide or increase affinity of the polypeptide for itsappropriate receptor, ligand and/or binding proteins. In some cases, theother additions, substitutions or deletions may increase the solubility(including but not limited to, when expressed in E. coli or other hostcells) of the polypeptide. In some embodiments sites are selected forsubstitution with a naturally encoded or non-natural amino acid inaddition to another site for incorporation of a non-natural amino acidfor the purpose of increasing the polypeptide solubility followingexpression in E. coli, or other recombinant host cells. In someembodiments, the polypeptides comprise another addition, substitution,or deletion that modulates affinity for the associated ligand, bindingproteins, and/or receptor, modulates (including but not limited to,increases or decreases) receptor dimerization, stabilizes receptordimers, modulates circulating half-life, modulates release orbio-availability, facilitates purification, or improves or alters aparticular route of administration. Similarly, the non-natural aminoacid polypeptide can comprise chemical or enzyme cleavage sequences,protease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including but not limited to, biotin) that improvedetection (including but not limited to, GFP), purification, transportthru tissues or cell membranes, prodrug release or activation, sizereduction, or other traits of the polypeptide.

IV. Growth Hormone Supergene Family as Exemplar

The methods, compositions, strategies and techniques described hereinare not limited to a particular type, class or family of polypeptides orproteins. Indeed, virtually any polypeptides may be designed or modifiedto include at least one “modified or unmodified” non-natural amino acidsdescribed herein. By way of example only, the polypeptide can behomologous to a therapeutic protein selected from the group consistingof: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody,antibody fragment, apolipoprotein, apoprotein, atrial natriureticfactor, atrial natriuretic polypeptide, atrial peptide, C—X—C chemokine,T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

Thus, the following description of the growth hormone (GH) supergenefamily is provided for illustrative purposes and by way of example only,and not as a limit on the scope of the methods, compositions, strategiesand techniques described herein. Further, reference to GH polypeptidesin this application is intended to use the generic term as an example ofany member of the GH supergene family. Thus, it is understood that themodifications and chemistries described herein with reference to GHpolypeptides or protein can be equally applied to any member of the GHsupergene family, including those specifically listed herein.

The following proteins include those encoded by genes of the growthhormone (GH) supergene family (Bazan, F., Immunology Today 11: 350-354(1990); Bazan, J. F. Science 257: 410-411 (1992); Mott, H. R. andCampbell, I. D., Current Opinion in Structural Biology 5: 114-121(1995); Silvennoinen, O. and Ihle, J. N., Signalling by theHematopoietic Cytokine Receptors (1996)): growth hormone, prolactin,placental lactogen, erythropoietin (EPO), thrombopoietin (TPO),interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11,IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophicfactor, leukemia inhibitory factor, alpha interferon, beta interferon,epsilon interferon, gamma interferon, omega interferon, tau interferon,granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF) and cardiotrophin-1 (CT-1) (“the GH supergene family”). It isanticipated that additional members of this gene family will beidentified in the future through gene cloning and sequencing. Members ofthe GH supergene family have similar secondary and tertiary structures,despite the fact that they generally have limited amino acid or DNAsequence identity. The shared structural features allow new members ofthe gene family to be readily identified and the non-natural amino acidmethods and compositions described herein similarly applied.

Structures of a number of cytokines, including G-CSF (Zink et al., FEBSLett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill etal., Proc. Natl. Acad. Sci. USA 90:5167 (1993)), GM-CSF (Diederichs, K.,et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol.224:1075-1085 (1992)), IL-2 (Bazan, J. F. and McKay, D. B., Science 257:410-413 (1992); IL-4 (Redfield et al., Biochemistry 30: 11029-11035(1991); Powers et al., Science 256:1673-1677 (1992)), and IL-5 (Milburnet al., Nature 363: 172-176 (1993)) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. IFNis considered to be a member of this family based upon modeling andother studies (Lee et al., J. Interferon Cytokine Res. 15:341 (1995);Murgolo et al., Proteins 17:62 (1993); Radhakrishnan et al., Structure4:1453 (1996); Klaus et al., J. Mol. Biol. 274:661 (1997)). A largenumber of additional cytokines and growth factors including ciliaryneurotrophic factor (CNTF), leukemia inhibitory factor (LIF),thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor(M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, andgranulocyte-colony stimulating factor (G-CSF), as well as the IFN's suchas alpha, beta, omega, tau, epsilon, and gamma interferon belong to thisfamily (reviewed in Mott and Campbell, Current Opinion in StructuralBiology 5: 114-121 (1995); Silvennoinen and Ihle (1996) Signalling bythe Hematopoietic Cytokine Receptors). All of the above cytokines andgrowth factors are now considered to comprise one large gene family.

In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, including but not limited to; GH and EPO, bind asingle type of receptor and cause it to form homodimers. Other familymembers, including but not limited to, IL-2, IL4, and IL-6, bind morethan one type of receptor and cause the receptors to form heterodimersor higher order aggregates (Davis et al., (1993) Science 260: 1805-1808;Paonessa et al., 1995) EMBO J. 14: 1942-1951; Mon and Campbell, CurrentOpinion in Structural Biology 5: 114-121 (1995)). Mutagenesis studieshave shown that, like GH, these other cytokines and growth factorscontain multiple receptor binding sites, typically two, and bind theircognate receptors sequentially (Mon and Campbell, Current Opinion inStructural Biology 5: 114-121 (1995); Matthews et al., (1996) Proc.Natl. Acad. Sci. USA 93: 9471-9476). Like GH, the primary receptorbinding sites for these other family members occur primarily in the fouralpha helices and the A-B loop. The specific amino acids in the helicalbundles that participate in receptor binding differ amongst the familymembers. Most of the cell surface receptors that interact with membersof the GH supergene family are structurally related and comprise asecond large multi-gene family. See, e.g. U.S. Pat. No. 6,608,183, whichis herein incorporated by reference in its entirety.

A general conclusion reached from mutational studies of various membersof the GH supergene family is that the loops joining the alpha helicesgenerally tend to not be involved in receptor binding. In particular theshort B-C loop appears to be non-essential for receptor binding in most,if not all, family members. For this reason, the B-C loop may besubstituted with non-natural amino acids as described herein in membersof the GH supergene family. The A-B loop, the C-D loop (and D-E loop ofinterferon/IL-10-like members of the GH superfamily) may also besubstituted with a non-natural amino acid. Amino acids proximal to helixA and distal to the final helix also tend not to be involved in receptorbinding and also may be sites for introducing non-natural amino acids.In some embodiments, a non-natural amino acid is substituted at anyposition within a loop structure including but not limited to the first1, 2, 3, 4, 5, 6, 7, or more amino acids of the A-B, B-C, C-D or D-Eloop. In some embodiments, a non-natural amino acid is substitutedwithin the last 1, 2, 3, 4, 5, 6, 7, or more amino acids of the A-B,B-C, C-D or D-E loop.

Certain members of the GH family, including but not limited to, EPO,IL-2, IL-3, IL-4, IL-6, IFN, GM-CSF, TPO, IL-10, IL-12 p35, IL-13, IL-15and beta interferon contain N-linked and/or O-linked sugars. Theglycosylation sites in the proteins occur almost exclusively in the loopregions and not in the alpha helical bundles. Because the loop regionsgenerally are not involved in receptor binding and because they aresites for the covalent attachment of sugar groups, they may be usefulsites for introducing non-natural amino acid substitutions into theproteins. Amino acids that comprise the N- and O-linked glycosylationsites in the proteins may be sites for non-natural amino acidsubstitutions because these amino acids are surface-exposed. Therefore,the natural protein can tolerate bulky sugar groups attached to theproteins at these sites and the glycosylation sites tend to be locatedaway from the receptor binding sites.

Additional members of the GH gene family are likely to be discovered inthe future. New members of the GH supergene family can be identifiedthrough computer-aided secondary and tertiary structure analyses of thepredicted protein sequences, and by selection techniques designed toidentify molecules that bind to a particular target. Members of the GHsupergene family typically possess four or five amphipathic helicesjoined by non-helical amino acids (the loop regions). The proteins maycontain a hydrophobic signal sequence at their N-terminus to promotesecretion from the cell. Such later discovered members of the GHsupergene family also are included within the methods and compositionsdescribed herein.

V. Non-Natural Amino Acids

The non-natural amino acids used in the methods and compositionsdescribed herein have at least one of the following four properties: (1)at least one functional group on the sidechain of the non-natural aminoacid has at least one characteristics and/or activity and/or reactivityorthogonal to the chemical reactivity of the 20 common,genetically-encoded amino acids (i.e., alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine), or at least orthogonal tothe chemical reactivity of the naturally occurring amino acids presentin the polypeptide that includes the non-natural amino acid; (2) theintroduced non-natural amino acids are substantially chemically inerttoward the 20 common, genetically-encoded amino acids; (3) thenon-natural amino acid can be stably incorporated into a polypeptide,preferably with the stability commensurate with the naturally-occurringamino acids or under typical physiological conditions, and furtherpreferably such incorporation can occur via an in vivo system; and (4)the non-natural amino acid includes an oxime functional group or afunctional group that can be transformed into an oxime group by reactingwith a reagent, preferably under conditions that do not destroy thebiological properties of the polypeptide that includes the non-naturalamino acid (unless of course such a destruction of biological propertiesis the purpose of the modification/transformation), or where thetransformation can occur under aqueous conditions at a pH between about4 and about 8, or where the reactive site on the non-natural amino acidis an electrophilic site. Illustrative, non-limiting examples of aminoacids that satisfy these four properties for non-natural amino acidsthat can be used with the compositions and methods described herein arepresented in FIGS. 2, 3, 35 and 40-43. Any number of non-natural aminoacids can be introduced into the polypeptide. Non-natural amino acidsmay also include protected or masked oximes or protected or maskedgroups that can be transformed into an oxime group after deprotection ofthe protected group or unmasking of the masked group. Non-natural aminoacids may also include protected or masked carbonyl or dicarbonylgroups, which can be transformed into a carbonyl or dicarbonyl groupafter deprotection of the protected group or unmasking of the maskedgroup and thereby are available to react with hydroxylamines or oximesto form oxime groups.

Non-natural amino acids that may be used in the methods and compositionsdescribed herein include, but are not limited to, amino acids comprisinga photoactivatable cross-linker, spin-labeled amino acids, fluorescentamino acids, metal binding amino acids, metal-containing amino acids,radioactive amino acids, amino acids with novel functional groups, aminoacids that covalently or noncovalently interact with other molecules,photocaged and/or photoisomerizable amino acids, amino acids comprisingbiotin or a biotin analogue, glycosylated amino acids such as a sugarsubstituted serine, other carbohydrate modified amino acids,keto-containing amino acids, aldehyde-containing amino acids, aminoacids comprising polyethylene glycol or other polyethers, heavy atomsubstituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

In some embodiments, non-natural amino acids comprise a saccharidemoiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

The chemical moieties incorporated into polypeptides via incorporationof non-natural amino acids into such polypeptides offer a variety ofadvantages and manipulations of polypeptides. For example, the uniquereactivity of a carbonyl or dicarbonyl functional group (including aketo- or aldehyde-functional group) allows selective modification ofproteins with any of a number of hydrazine- or hydroxylamine-containingreagents in vivo and in vitro. A heavy atom non-natural amino acid, forexample, can be useful for phasing x-ray structure data. Thesite-specific introduction of heavy atoms using non-natural amino acidsalso provides selectivity and flexibility in choosing positions forheavy atoms. Photoreactive non-natural amino acids (including but notlimited to, amino acids with benzophenone and arylazides (including butnot limited to, phenylazide) side chains), for example, allow forefficient in vivo and in vitro photocrosslinking of polypeptides.Examples of photoreactive non-natural amino acids include, but are notlimited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Thepolypeptide with the photoreactive non-natural amino acids may then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In a non-limiting example, the methyl group of anon-natural amino can be substituted with an isotopically labeled,including but not limited to, with a methyl group, as a probe of localstructure and dynamics, including but not limited to, with the use ofnuclear magnetic resonance and vibrational spectroscopy.

A. Structure and Synthesis of Non-Natural Amino Acids: Carbonyl,Carbonyl like, Masked Carbonyl, and Protected Carbonyl Groups

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via various chemical reactions, including,but not limited to, nucleophilic addition reactions. Such electrophilicreactive groups include a carbonyl- or dicarbonyl-group (including aketo- or aldehyde group), a carbonyl-like- or dicarbonyl-like-group(which has reactivity similar to a carbonyl- or dicarbonyl-group and isstructurally similar to a carbonyl- or dicarbonyl-group), a maskedcarbonyl- or masked dicarbonyl-group (which can be readily convertedinto a carbonyl- or dicarbonyl-group), or a protected carbonyl- orprotected dicarbonyl-group (which has reactivity similar to a carbonyl-or dicarbonyl-group upon deprotection). Such amino acids include aminoacids having the structure of Formula (I):

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   J is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   each R″ is independently H, alkyl, substituted alkyl, or a    protecting group, or when more than one R″ group is present, two R″    optionally form a heterocycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   or the -A-B-J-R groups together form a bicyclic or tricyclic    cycloalkyl or heterocycloalkyl comprising at least one carbonyl    group, including a dicarbonyl group, protected carbonyl group,    including a protected dicarbonyl group, or masked carbonyl group,    including a masked dicarbonyl group;-   or the -J-R group together forms a monocyclic or bicyclic cycloalkyl    or heterocycloalkyl comprising at least one carbonyl group,    including a dicarbonyl group, protected carbonyl group, including a    protected dicarbonyl group, or masked carbonyl group, including a    masked dicarbonyl group; with a proviso that when A is phenylene and    each R₃ is H, B is present; and that when A is —(CH₂)₄— and each R₃    is H, B is not —NHC(O)(CH₂CH₂)—; and that when A and B are absent    and each R₃ is H, R is not methyl. Such non-natural amino acids may    be in the form of a salt, or may be incorporated into a non-natural    amino acid polypeptide, polymer, polysaccharide, or a polynucleotide    and optionally post translationally modified.

In certain embodiments, compounds of Formula (I) are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In certainembodiments, compounds of Formula (I) are stable for at least 2 weeksunder mildly acidic conditions. In certain embodiments, compound ofFormula (I) are stable for at least 5 days under mildly acidicconditions. In certain embodiments, such acidic conditions are pH 2 to8.

In certain embodiments of compounds of Formula (I), B is lower alkylene,substituted lower alkylene, —O-(alkylene or substituted alkylene)-,—C(R′)═N—N(R′)—, —N(R′)CO—, —C(O)—, —C(R′)═N—, —C(O)-(alkylene orsubstituted alkylene)-, —CON(R′)-(alkylene or substituted alkylene)-,—S(alkylene or substituted alkylene)-, —S(O)(alkylene or substitutedalkylene)-, or —S(O)₂(alkylene or substituted alkylene)-. In certainembodiments of compounds of Formula (I), B is —O(CH₂)—, —CH═N—,—CH═N—NH—, —NHCH₂—, —NHCO—, —C(O)—, —C(O)—(CH₂)—, —CONH—(CH₂)—, —SCH₂—,—S(═O)CH₂—, or —S(O)₂CH₂—. In certain embodiments of compounds ofFormula (I), R is C₁₋₆ alkyl or cycloalkyl. In certain embodiments ofcompounds of Formula (I) R is CH₃, —CH(CH₃)₂, or cyclopropyl. In certainembodiments of compounds of Formula (I), R₁ is H, tert-butyloxycarbonyl(Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl(TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compoundsof Formula (I), R₁ is a resin, amino acid, polypeptide, orpolynucleotide. In certain embodiments of compounds of Formula (I), R₂is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments ofcompounds of Formula (I), R₂ is a resin, amino acid, polypeptide, orpolynucleotide. In certain embodiments of compounds of Formula (I), R₂is a polynucleotide. In certain embodiments of compounds of Formula (I),R₂ is ribonucleic acid (RNA). In certain embodiments of compounds ofFormula (I), R₂ is tRNA. In certain embodiments of compounds of Formula(I), the tRNA specifically recognizes a selector codon. In certainembodiments of compounds of Formula (I) the selector codon is selectedfrom the group consisting of an amber codon, ochre codon, opal codon, aunique codon, a rare codon, an unnatural codon, a five-base codon, and afour-base codon. In certain embodiments of compounds of Formula (I), R₂is a suppressor tRNA.

In certain embodiments of compounds of Formula (I),

is selected from the group consisting of:

-   -   (i) A is substituted lower alkylene, C₄-arylene, substituted        arylene, heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a divalent linker selected        from the group consisting of lower alkylene, substituted lower        alkylene, lower alkenylene, substituted lower alkenylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S(O)—, —S(O)₂—,        —NS(O)₂—, —OS(O)₂—, —C(O)—, —C(O)-(alkylene or substituted        alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or        substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or        substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′),        —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,        —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—,        —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—;    -   (ii) A is optional, and when present is substituted lower        alkylene, C₄-arylene, substituted arylene, heteroarylene,        substituted heteroarylene, alkarylene, substituted alkarylene,        aralkylene, or substituted aralkylene;        -   B is a divalent linker selected from the group consisting of            lower alkylene, substituted lower alkylene, lower            alkenylene, substituted lower alkenylene, —O—, —O-(alkylene            or substituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—,            —OS(O)₂—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,            —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or            substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or            substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,            —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—;    -   (iii) A is lower alkylene;        -   B is optional, and when present is a divalent linker            selected from the group consisting of lower alkylene,            substituted lower alkylene, lower alkenylene, substituted            lower alkenylene, —O—, —O-(alkylene or substituted            alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—, —OS(O)₂—,            —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,            —N(R′)—, —C(O)N(R′)—, —CSN(R′)—, —CON(R′)-(alkylene or            substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,            —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—; and    -   (iv) A is phenylene;        -   B is a divalent linker selected from the group consisting of            lower alkylene, substituted lower alkylene, lower            alkenylene, substituted lower alkenylene, —O—, —O-(alkylene            or substituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—,            —OS(O)₂—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,            —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or            substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or            substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,            —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—;    -   J is

-   -   each R′ is independently H, alkyl, or substituted alkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   each R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;

In addition, amino acids having the structure of Formula (II) areincluded:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;    with a proviso that when A is phenylene, B is present; and that when    A is (CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B are    absent, R is not methyl. Such non-natural amino acids may be in the    form of a salt, or may be incorporated into a non-natural amino acid    polypeptide, polymer, polysaccharide, or a polynucleotide and    optionally post translationally modified.

In addition, amino acids having the structure of Formula (III) areincluded:

wherein:

-   B is a linker selected from the group consisting of lower alkylene,    substituted lower alkylene, lower alkenylene, substituted lower    alkenylene, lower heteroalkylene, substituted lower heteroalkylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,    —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl or substituted alkyl;-   R is H, alkyl substituted alkyl cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl substituted alkyl —N(R′)₂, —C(O)_(k)R′ where k is 1,    2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl or substituted alkyl Such non-natural amino    acids may be in the form of a salt, or may be incorporated into a    non-natural amino acid polypeptide, polymer, polysaccharide, or a    polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included:

Such non-natural amino acids may be are optionally amino protectedgroup, carboxyl protected and/or in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids having the structure of Formula(IV) are included:

wherein

-   —NS(O)₂—, —OS(O)₂—, optional, and when present is a linker selected    from the group consisting of lower alkylene, substituted lower    alkylene, lower alkenylene, substituted lower alkenylene, lower    heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene    or substituted alkylene)-, —S—, —S-(alkylene or substituted    alkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or    substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8;    with a proviso that when A is (CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

-   -   wherein such compounds are optionally amino protected,        optionally carboxyl protected, optionally amino protected and        carboxyl protected, or a salt thereof, or may be incorporated        into a non-natural amino acid polypeptide, polymer,        polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein,

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(IX) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;

wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups. For example, thefollowing amino acids having the structure of Formula (V) are included:

wherein,

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(VI) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;    -   wherein each R_(a) is independently selected from the group        consisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂,        —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and        —S(O)_(k)R′, where each R′ is independently H, alkyl, or        substituted alkyl.        Such non-natural amino acids may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxylprotected, or a salt thereof. Such non-natural amino acids may be in theform of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(VII) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;    -   each R_(a) is independently selected from the group consisting        of H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′        where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where        each R′ is independently H, alkyl, or substituted alkyl; and n        is 0 to 8.        Such non-natural amino acids may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxylprotected, or a salt thereof, or may be incorporated into a non-naturalamino acid polypeptide, polymer, polysaccharide, or a polynucleotide andoptionally post translationally modified.

In addition, the following amino acids having the structure of Formula(XXX) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,    alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXX-A) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXX-B) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXI) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and    R⁹ on each CR⁸R⁹ group is independently selected from the group    consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸    and R⁹ can together form ═O or a cycloalkyl, or any to adjacent R⁸    groups can together form a cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXI-A) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;    -   n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group        is independently selected from the group consisting of H,        alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can        together form ═O or a cycloalkyl, or any to adjacent R⁸ groups        can together form a cycloalkyl.        Such non-natural amino acids may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, the following amino acids having the structure of Formula(XXXI-B) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;    -   n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group        is independently selected from the group consisting of H,        alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can        together form ═O or a cycloalkyl, or any to adjacent R⁸ groups        can together form a cycloalkyl.        Such non-natural amino acids may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, the following amino acids having the structure of Formula(XXXII) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,    alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

The In addition, the following amino acids having the structure ofFormula (XXXII-A) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXII-B) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, amino acids having the structure of Formula (XXXX) areincluded:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene; M is —C(R₃)—,

where (a) indicates bonding to

-   -   the A group and (b) indicates bonding to respective carbonyl        groups, R₃ and R₄ are independently chosen from H, halogen,        alkyl substituted alkyl cycloalkyl, or substituted cycloalkyl,        or R₃ and R₄ or two R₃ groups or two R₄ groups optionally form a        cycloalkyl or a heterocycloalkyl;        R is H, halogen, alkyl substituted alkyl cycloalkyl, or        substituted cycloalkyl;        T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl        substituted alkyl cycloalkyl, or substituted cycloalkyl;        R₁ is H, an amino protecting group, resin, amino acid,        polypeptide, or polynucleotide; and        R₂ is OH, an ester protecting group, resin, amino acid,        polypeptide, or polynucleotide.        Such non-natural amino acids may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, amino acids having the structure of Formula (XXXXI) areincluded:

wherein:

M is —C(R₃)—,

where (a) indicates bonding to

-   -   the A group and (b) indicates bonding to respective carbonyl        groups, R₃ and R₄ are independently chosen from H, halogen,        alkyl substituted alkyl cycloalkyl, or substituted cycloalkyl,        or R₃ and R₄ or two R₃ groups or two R₄ groups optionally form a        cycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl substituted alkyl cycloalkyl, or substituted    cycloalkyl;

-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl    substituted alkyl cycloalkyl, or substituted cycloalkyl;

-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and

-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;    -   each R_(a) is independently selected from the group consisting        of H, halogen, alkyl substituted alkyl, —N(R′)₂, —C(O)_(k)R′        where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where        each R′ is independently H, alkyl or substituted alkyl.        Such non-natural amino acids may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In addition, amino acids having the structure of Formula (XXXXII) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; and T₃ is O, or S.Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, amino acids having the structure of Formula (XXXXIII) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl.

In addition, the following amino acids having structures of Formula(XXXXIII) are included:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117(14):3893-3899 (1995). Moreover, the unique reactivity of thecarbonyl or dicarbonyl group allows for selective modification in thepresence of the other amino acid side chains. See, e.g., Cornish, V. W.,et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. &Stroh, J. G., Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al.,Science 276:1125-1128 (1997).

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), incorporated by reference. Othercarbonyl- or dicarbonyl-containing amino acids can be similarlyprepared. Further, non-limiting exemplary syntheses of non-natural aminoacid that are include herein are presented in FIGS. 4, 24-34 and 36-39.

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

Additionally, by way of example a non-natural amino acid bearingadjacent hydroxyl and amino groups can be incorporated into apolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685.

B. Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

Non-natural amino acids containing a hydroxylamine (also called anaminooxy) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain carbonyl- or dicarbonyl-groups, including but notlimited to, ketones, aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9):727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl- ordicarbonyl-containing group such as, by way of example, a ketones,aldehydes or other functional groups with similar chemical reactivity.

Thus, in certain embodiments described herein are non-natural aminoacids with sidechains comprising a hydroxylamine group, ahydroxylamine-like group (which has reactivity similar to ahydroxylamine group and is structurally similar to a hydroxylaminegroup), a masked hydroxylamine group (which can be readily convertedinto a hydroxylamine group), or a protected hydroxylamine group (whichhas reactivity similar to a hydroxylamine group upon deprotection). Suchamino acids include amino acids having the structure of Formula:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   K is —NR₆R₇ or N═CR₆R₇;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and-   L is optional, and when present is a linker selected from the group    consisting of alkylene, substituted alkylene, alkenylene,    substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,    —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is    1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In certain embodiments of compounds of Formula (XIV), A is phenylene orsubstituted phenylene. In certain embodiments of compounds of Formula(XIV), B is -(alkylene or substituted alkylene)-, —O-(alkylene orsubstituted alkylene)-, —S-(alkylene or substituted alkylene)-, or—C(O)-(alkylene or substituted alkylene)-. In certain embodiments ofcompounds of Formula (XIV), B is O(CH₂)₂—, S(CH₂)₂—, NH(CH₂)₂—,CO(CH₂)₂—, or —(CH₂)_(n)— where n is 1 to 4. In certain embodiments ofcompounds of Formula (XIV), R₁ is H, tert-butyloxycarbonyl (Boc),9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), orbenzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula(XIV), wherein R₁ is a resin, amino acid, polypeptide, orpolynucleotide. In certain embodiments of compounds of Formula (XIV),wherein R₂ is OH, O-methyl, O-ethyl, or O-t-butyl. In certainembodiments of compounds of Formula (XIV), wherein R₂ is a resin, aminoacid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (XIV), wherein R₂ is a polynucleotide. In certainembodiments of compounds of Formula (XIV), wherein R₂ is ribonucleicacid (RNA). In certain embodiments of compounds of Formula (XIV),wherein R₂ is tRNA. In certain embodiments of compounds of Formula(XIV), wherein the tRNA specifically recognizes a selector codon. Incertain embodiments of compounds of Formula (XIV), wherein the selectorcodon is selected from the group consisting of an amber codon, ochrecodon, opal codon, a unique codon, a rare codon, an unnatural codon, afive-base codon, and a four-base codon. In certain embodiments ofcompounds of Formula (XIV), wherein R₂ is a suppressor tRNA. In certainembodiments of compounds of Formula (XIV), each of R₆ and R₇ isindependently selected from the group consisting of H, alkyl,substituted alkyl, alkoxy, substituted alkoxy, polyalkylene oxide,substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, andsubstituted aralkyl. In certain embodiments of compounds of Formula(XIV), each of R₆ and R₇ is independently selected from the groupconsisting of H, methyl, phenyl, and [(alkylene or substitutedalkylene)-O-(hydrogen, alkyl, or substituted alkyl)]_(x), wherein x isfrom 1-50. In certain embodiments of compounds of Formula (XIV), K is—NR₆R₇.

In certain embodiments of compounds of Formula (XIV), X is abiologically active agent selected from the group consisting of apeptide, protein, enzyme, antibody, drug, dye, lipid, nucleosides,oligonucleotide, cell, virus, liposome, microparticle, and micelle. Incertain embodiments of compounds of Formula (XIV), X is a drug selectedfrom the group consisting of an antibiotic, fungicide, anti-viral agent,anti-inflammatory agent, anti-tumor agent, cardiovascular agent,anti-anxiety agent, hormone, growth factor, and steroidal agent. Incertain embodiments of compounds of Formula (XIV), X is an enzymeselected from the group consisting of horseradish peroxidase, alkalinephosphatase, β-galactosidase, and glucose oxidase. In certainembodiments of compounds of Formula (XIV), X is a detectable labelselected from the group consisting of a fluorescent, phosphorescent,chemiluminescent, chelating, electron dense, magnetic, intercalating,radioactive, chromophoric, and energy transfer moiety.

In certain embodiments, compounds of Formula (XIV) are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In certainembodiments, compounds of Formula (XIV) are stable for at least 2 weeksunder mildly acidic conditions. In certain embodiments, compound ofFormula (XIV) are stable for at least 5 days under mildly acidicconditions. In certain embodiments, such acidic conditions are pH 2 to8.

Such amino acids include amino acids having the structure of Formula(XV):

wherein

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

A non-limiting, representative amino acid has the following structure:

Such a non-natural amino acid may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G. et al.,Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acidscan be similarly prepared. Further, non-limiting exemplary synthesis ofa non-natural amino acid described herein are presented in FIG. 5.

C. Chemical Synthesis of Non-Natural Amino Acids: Oxime-Containing AminoAcids

Non-natural amino acids containing an oxime group allow for reactionwith a variety of reagents that contain certain reactive carbonyl- ordicarbonyl-groups (including but not limited to, ketones, aldehydes, orother groups with similar reactivity) to form new non-natural aminoacids comprising a new oxime group. Such an oxime exchange reactionallow for the further functionalization of non-natural amino acidpolypeptides. Further, the original non-natural amino acids containingan oxime group may be useful in their own right as long as the oximelinkage is stable under conditions necessary to incorporate the aminoacid into a polypeptide (e.g., the in vivo, in vitro and chemicalsynthetic methods described herein).

Thus, in certain embodiments described herein are non-natural aminoacids with sidechains comprising an oxime group, an oxime-like group(which has reactivity similar to an oxime group and is structurallysimilar to an oxime group), a masked oxime group (which can be readilyconverted into an oxime group), or a protected oxime group (which hasreactivity similar to an oxime group upon deprotection). Such aminoacids include amino acids having the structure of Formula (XI):

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   R₅ is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,    substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,    substituted aralkyl, -(alkylene or substituted alkylene)-ON(R″)₂,    -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or    substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″,    —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independently hydrogen,    alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,    substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,    substituted alkaryl, aralkyl, or substituted aralkyl;-   or R₅ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, -(alkylene or substituted    alkylene)-O—N═CR′-, -(alkylene or substituted    alkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene or    substituted alkylene)-S(O)_(k)-(alkylene or substituted    alkylene)-S—, -(alkylene or substituted alkylene)-S—S—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;    with a proviso that when A and B are absent, R is not methyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In certain embodiments of compounds of Formula (XI), B is —O-(alkyleneor substituted alkylene)-. In certain embodiments of compounds ofFormula (XI), B is O(CH₂)—. In certain embodiments of compounds ofFormula (XI), R is C₁₋₆ alkyl. In certain embodiments of compounds ofFormula (XI), R is —CH₃. In certain embodiments of compounds of Formula(XI), R₁ is H, tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl(Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz).In certain embodiments of compounds of Formula (XI), R₁ is a resin,amino acid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (XI), R₂ is OH, O-methyl, O-ethyl, or O-t-butyl. Incertain embodiments of compounds of Formula (XI), R₂ is a resin, aminoacid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (XI), R₂ is a polynucleotide. In certainembodiments of compounds of Formula (XI), R₂ is ribonucleic acid (RNA).In certain embodiments of compounds of Formula (XI), R₂ is tRNA. Incertain embodiments of compounds of Formula (XI), the tRNA specificallyrecognizes a selector codon. In certain embodiments of compounds ofFormula (XI), the selector codon is selected from the group consistingof an amber codon, ochre codon, opal codon, a unique codon, a rarecodon, an unnatural codon, a five-base codon, and a four-base codon. Incertain embodiments of compounds of Formula (XI), R₂ is a suppressortRNA. In certain embodiments of compounds of Formula (XI), R₅ isalkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substitutedpolyalkylene oxide, or —C(O)₂R″. In certain embodiments of compounds ofFormula (XI), R₅ is [(alkylene or substituted alkylene)-O-(hydrogen,alkyl, or substituted alkyl)]_(x), wherein x is from 1-50. In certainembodiments of compounds of Formula (XI), R₅ is (CH₂CH₂)—O—CH₃ or —COOH.

In certain embodiments, compounds of Formula (I) are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In certainembodiments, compounds of Formula (I) are stable for at least 2 weeksunder mildly acidic conditions. In certain embodiments, compound ofFormula (I) are stable for at least 5 days under mildly acidicconditions. In certain embodiments, such acidic conditions are pH 2 to8.

Amino acids of Formula (XI) include amino acids having the structure ofFormula (XII):

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   R₅ is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,    substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,    substituted aralkyl, -(alkylene or substituted alkylene)-ON(R″)₂,    -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or    substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″,    —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independently hydrogen,    alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,    substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,    substituted alkaryl, aralkyl, or substituted aralkyl;-   or R₅ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, -(alkylene or substituted    alkylene)-O—N═CR′—, -(alkylene or substituted    alkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene or    substituted alkylene)-S(O)_(k)-(alkylene or substituted    alkylene)-S—, -(alkylene or substituted alkylene)-S—S—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

Such amino acids include amino acids having the structure of Formula(XIII):

wherein,

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   R₅ is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,    substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,    substituted aralkyl, -(alkylene or substituted alkylene)-ON(R″)₂,    -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or    substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″,    —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independently hydrogen,    alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,    substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,    substituted alkaryl, aralkyl, or substituted aralkyl;-   or R₅ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, -(alkylene or substituted    alkylene)-O—N═CR′—, -(alkylene or substituted    alkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene or    substituted alkylene)-S(O)_(k)-(alkylene or substituted    alkylene)-S—, -(alkylene or substituted alkylene)-S—S—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

Further non-limiting examples of such amino acids include amino acidshaving the following structures:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, such amino acids include amino acids having the structureof Formula (XIV):

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   K is —NR₆R₇ or N═CR₆R₇;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

Such amino acids further include amino acids having the structure ofFormula (XVI):

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

Further, such amino acids include amino acids having the structure ofFormula (XVII):

wherein:

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl.

Non-limiting examples of such amino acids include amino acids having thefollowing structures:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Additionally, such amino acids include amino acids having the structureof Formula (XVIII):

wherein:

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; a    photoisomerizable moiety; biotin; a biotin analogue; a moiety    incorporating a heavy atom; a chemically cleavable group; a    photocleavable group; an elongated side chain; a carbon-linked    sugar; a redox-active agent; an amino thioacid; a toxic moiety; an    isotopically labeled moiety; a biophysical probe; a phosphorescent    group; a chemiluminescent group; an electron dense group; a magnetic    group; an intercalating group; a chromophore; an energy transfer    agent; a biologically active agent; a detectable label; and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl; and-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and S(O)_(k)R′; where each R′ is    independently H, alkyl, or substituted alkyl and n is 0 to 8.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

Non-limiting examples of such amino acids include amino acids having thefollowing structures:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Oxime-based non-natural amino acids may be synthesized by methodsalready described in the art, or by methods described herein, including:(a) reaction of a hydroxylamine-containing non-natural amino acid with acarbonyl- or dicarbonyl-containing reagent; (b) reaction of a carbonyl-or dicarbonyl-containing non-natural amino acid with ahydroxylamine-containing reagent; or (c) reaction of an oxime-containingnon-natural amino acid with certain carbonyl- or dicarbonyl-containingreagents, including by way of example, a ketone-containing reagent or analdehyde-containing reagent. FIGS. 5 and 6 present representative,non-limiting examples of these synthetic methodologies.

D. Cellular Uptake of Non-Natural Amino Acids

Non-natural amino acid uptake by a eukaryotic cell is one issue that istypically considered when designing and selecting non-natural aminoacids, including but not limited to, for incorporation into a protein.For example, the high charge density of α-amino acids suggests thatthese compounds are unlikely to be cell permeable. Natural amino acidsare taken up into the eukaryotic cell via a collection of protein-basedtransport systems. A rapid screen can be done which assesses whichnon-natural amino acids, if any, are taken up by cells (examples 15 & 16herein illustrate non-limiting examples of tests which can be done onnon-natural amino acids). See, e.g., the toxicity assays in, e.g., theU.S. Patent Publication No. 2004/198637 entitled “Protein Arrays,” whichis herein incorporated by reference in its entirety, and Liu, D. R. &Schultz, P. G. (1999) Progress toward the evolution of an organism withan expanded genetic code. PNAS United States 96:4780-4785. Althoughuptake is easily analyzed with various assays, an alternative todesigning non-natural amino acids that are amenable to cellular uptakepathways is to provide biosynthetic pathways to create amino acids invivo.

Typically, the non-natural amino acid produced via cellular uptake asdescribed herein is produced in a concentration sufficient for efficientprotein biosynthesis, including but not limited to, a natural cellularamount, but not to such a degree as to affect the concentration of theother amino acids or exhaust cellular resources. Typical concentrationsproduced in this manner are about 10 mM to about 0.05 mM.

E. Biosynthesis of Non-Natural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular non-natural amino acid may not exist in nature, including butnot limited to, in a cell, the methods and compositions described hereinprovide such methods. For example, biosynthetic pathways for non-naturalamino acids can be generated in host cell by adding new enzymes ormodifying existing host cell pathways. Additional new enzymes includenaturally occurring enzymes or artificially evolved enzymes. Forexample, the biosynthesis of p-aminophenylalanine (as presented in anexample in WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids”) relies on the addition of a combination of known enzymesfrom other organisms. The genes for these enzymes can be introduced intoa eukaryotic cell by transforming the cell with a plasmid comprising thegenes. The genes, when expressed in the cell, provide an enzymaticpathway to synthesize the desired compound. Examples of the types ofenzymes that are optionally added are provided herein. Additionalenzymes sequences are found, for example, in Genbank. Artificiallyevolved enzymes can be added into a cell in the same manner. In thismanner, the cellular machinery and resources of a cell are manipulatedto produce non-natural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the world wide web atwww.maxygen.com), can be used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the worldwide web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate a non-natural amino acid in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to those identified through functionalgenomics, molecular evolution and design. Diversa Corporation (availableon the world wide web at diversa.com) also provides technology forrapidly screening libraries of genes and gene pathways, including butnot limited to, to create new pathways for biosynthetically producingnon-natural amino acids.

Typically, the non-natural amino acid produced with an engineeredbiosynthetic pathway as described herein is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anon-natural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the non-natural amino acidfor both ribosomal protein synthesis and cell growth.

F. Additional Synthetic Methodology

The non-natural amino acids described herein may be synthesized usingmethodologies described in the art or using the techniques describedherein or by a combination thereof. As an aid, the following tableprovides various starting electrophiles and nucleophiles which may becombined to create a desired functional group. The information providedis meant to be illustrative and not limiting to the synthetic techniquesdescribed herein.

TABLE 1 Examples of Covalent Linkages and Precursors Thereof CovalentLinkage Product Electrophile Nucleophile Carboxamides Activated estersamines/anilines Carboxamides acyl azides amines/anilines Carboxamidesacyl halides amines/anilines Esters acyl halides alcohols/phenols Estersacyl nitriles alcohols/phenols Carboxamides acyl nitrilesamines/anilines Imines Aldehydes amines/anilines Hydrazones aldehydes orketones Hydrazines Oximes aldehydes or ketones Hydroxylamines Alkylamines alkyl halides amines/anilines Esters alkyl halides carboxylicacids Thioethers alkyl halides Thiols Ethers alkyl halidesalcohols/phenols Thioethers alkyl sulfonates Thiols Esters alkylsulfonates carboxylic acids Ethers alkyl sulfonates alcohols/phenolsEsters Anhydrides alcohols/phenols Carboxamides Anhydridesamines/anilines Thiophenols aryl halides Thiols Aryl amines aryl halidesAmines Thioethers Azindines Thiols Boronate esters Boronates GlycolsCarboxamides carboxylic acids amines/anilines Esters carboxylic acidsAlcohols hydrazines Hydrazides carboxylic acids N-acylureas orAnhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylicacids Thioethers Epoxides Thiols Thioethers haloacetamides ThiolsAmmotriazines halotriazines amines/anilines Triazinyl ethershalotriazines alcohols/phenols Amidines imido esters amines/anilinesUreas Isocyanates amines/anilines Urethanes Isocyanates alcohols/phenolsThioureas isothiocyanates amines/anilines Thioethers Maleimides ThiolsPhosphite esters phosphoramidites Alcohols Silyl ethers silyl halidesAlcohols Alkyl amines sulfonate esters amines/anilines Thioetherssulfonate esters Thiols Esters sulfonate esters carboxylic acids Etherssulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilinesSulfonate esters sulfonyl halides phenols/alcohols

In general, carbon electrophiles are susceptible to attack bycomplementary nucleophiles, including carbon nucleophiles, wherein anattacking nucleophile brings an electron pair to the carbon electrophilein order to form a new bond between the nucleophile and the carbonelectrophile.

Non-limiting examples of carbon nucleophiles include, but are notlimited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium,organozinc, alkyl-, alkenyl , aryl- and alkynyl-tin reagents(organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents(organoboranes and organoboronates); these carbon nucleophiles have theadvantage of being kinetically stable in water or polar organicsolvents. Other non-limiting examples of carbon nucleophiles includephosphorus ylids, enol and enolate reagents; these carbon nucleophileshave the advantage of being relatively easy to generate from precursorswell known to those skilled in the art of synthetic organic chemistry.Carbon nucleophiles, when used in conjunction with carbon electrophiles,engender new carbon-carbon bonds between the carbon nucleophile andcarbon electrophile.

Non-limiting examples of non-carbon nucleophiles suitable for couplingto carbon electrophiles include but are not limited to primary andsecondary amines, thiols, thiolates, and thioethers, alcohols,alkoxides, azides, semicarbazides, and the like. These non-carbonnucleophiles, when used in conjunction with carbon electrophiles,typically generate heteroatom linkages (C—X—C), wherein X is ahetereoatom, including, but not limited to, oxygen, sulfur, or nitrogen.

VI. Polypeptides with Non-Natural Amino Acids

For convenience, the form, properties and other characteristics of thecompounds described in this section have been described genericallyand/or with specific examples. However, the form, properties and othercharacteristics described in this section should not be limited to justthe generic descriptions or specific example provided in this section,but rather the form, properties and other characteristics described inthis section apply equally well to all compounds that fall within thescope of Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII, includingany sub-formulas or specific compounds that fall within the scope ofFormulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII that are described inthe specification, claims and figures herein.

The compositions and methods described herein provide for theincorporation of at least one non-natural amino acid into a polypeptide.The non-natural amino acid may be present at any location on thepolypeptide, including any terminal position or any internal position ofthe polypeptide. Preferably, the non-natural amino acid does not destroythe activity and/or the tertiary structure of the polypeptide relativeto the homologous naturally-occurring amino acid polypeptide, unlesssuch destruction of the activity and/or tertiary structure was one ofthe purposes of incorporating the non-natural amino acid into thepolypeptide. Further, the incorporation of the non-natural amino acidinto the polypeptide may modify to some extent the activity (e.g.,manipulating the therapeutic effectiveness of the polypeptide, improvingthe safety profile of the polypeptide, adjusting the pharmacokinetics,pharmacologics and/or pharmacodynamics of the polypeptide (e.g.,increasing water solubility, bioavailability, increasing serumhalf-life, increasing therapeutic half-life, modulating immunogenicity,modulating biological activity, or extending the circulation time),providing additional functionality to the polypeptide, incorporating atag, label or detectable signal into the polypeptide, easing theisolation properties of the polypeptide, and any combination of theaforementioned modifications) and/or tertiary structure of thepolypeptide relative to the homologous naturally-occurring amino acidpolypeptide without fully causing destruction of the activity and/ortertiary structure. Such modifications of the activity and/or tertiarystructure are often one of the goals of effecting such incorporations,although the incorporation of the non-natural amino acid into thepolypeptide may also have little effect on the activity and/or tertiarystructure of the polypeptide relative to the homologousnaturally-occurring amino acid polypeptide. Correspondingly, non-naturalamino acid polypeptides, compositions comprising non-natural amino acidpolypeptides, methods for making such polypeptides and polypeptidecompositions, methods for purifying, isolating, and characterizing suchpolypeptides and polypeptide compositions, and methods for using suchpolypeptides and polypeptide compositions are considered within thescope of the present disclosure. Further, the non-natural amino acidpolypeptides described herein may also be ligated to another polypeptide(including, by way of example, a non-natural amino acid polypeptide or anaturally-occurring amino acid polypeptide).

The non-natural amino acid polypeptides described herein may be producedbiosynthetically or non-biosynthetically. By biosynthetically is meantany method utilizing a translation system (cellular or non-cellular),including use of at least one of the following components: apolynucleotide, a codon, a tRNA, and a ribosome. By non-biosyntheticallyis meant any method not utilizing a translation system: this approachcan be further divided into methods utilizing solid state peptidesynthetic methods, solid phase peptide synthetic methods, methods thatutilize at least one enzyme, and methods that do not utilize at leastone enzyme; in addition any of this sub-divisions may overlap and manymethods may utilize a combination of these sub-divisions.

The methods, compositions, strategies and techniques described hereinare not limited to a particular type, class or family of polypeptides orproteins. Indeed, virtually any polypeptide may include at least onenon-natural amino acids described herein. By way of example only, thepolypeptide can be homologous to a therapeutic protein selected from thegroup consisting of: alpha-1 antitrypsin, angiostatin, antihemolyticfactor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor,atrial natriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765,NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4,MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone. In a related or further embodiment, the non-naturalamino acid polypeptide may also be homologous to any polypeptide memberof the growth hormone supergene family.

The non-natural amino acid polypeptides may be further modified asdescribed elsewhere in this disclosure or the non-natural amino acidpolypeptide may be used without further modification. Incorporation of anon-natural amino acid into a polypeptide can be done for a variety ofpurposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for apolypeptide array), etc. Polypeptides that include a non-natural aminoacid can have enhanced or even entirely new catalytic or biophysicalproperties. By way of example only, the following properties can bemodified by inclusion of a non-natural amino acid into a polypeptide:toxicity, biodistribution, structural properties, spectroscopicproperties, chemical and/or photochemical properties, catalytic ability,half-life (including but not limited to, serum half-life), ability toreact with other molecules, including but not limited to, covalently ornoncovalently, and the like. Compositions with polypeptides that includeat least one non-natural amino acid are useful for, including but notlimited to, novel therapeutics, diagnostics, catalytic enzymes,industrial enzymes, binding proteins (including but not limited to,antibodies), and research including, but not limited to, the study ofprotein structure and function. See, e.g., Dougherty, (2000) UnnaturalAmino Acids as Probes of Protein Structure and Function, Current Opinionin Chemical Biology, 4:645-652.

Further, the sidechain of the non-natural amino acid component(s) of apolypeptide can provide a wide range of additional functionality to thepolypeptide; by way of example only, and not as a limitation, thesidechain of the non-natural amino acid portion of a polypeptide mayinclude any of the following: a label; a dye; a polymer; a water-solublepolymer; a derivative of polyethylene glycol; a photocrosslinker; acytotoxic compound; a drug; an affinity label; a photoaffinity label; areactive compound; a resin; a second protein or polypeptide orpolypeptide analog; an antibody or antibody fragment; a metal chelator;a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; aRNA; an antisense polynucleotide; a saccharide, a water-solubledendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label;a fluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof.

In one aspect, a composition includes at least one polypeptide with atleast one, including but not limited to, at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, or at least ten or more non-natural amino acids.Such non-natural amino acids may be the same or different. In addition,there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more different sites in the polypeptide which comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore different, or the same, non-natural amino acids. In another aspect,a composition includes a polypeptide with at least one, but fewer thanall, of a particular amino acid present in the polypeptide issubstituted with a non-natural amino acid(s). For a given polypeptidewith more than one non-natural amino acids, the non-natural amino acidscan be identical or different (such as, by way of example only, thepolypeptide can include two or more different types of non-natural aminoacids, or can include two of the same non-natural amino acid). For agiven polypeptide with more than two non-natural amino acids, thenon-natural amino acids can be the same, different or a combination of amultiple number of non-natural amino acids of the same kind with atleast one different non-natural amino acid.

Although embodiments of the non-natural amino acid polypeptidesdescribed herein may be chemically synthesized via solid phase peptidesynthesis methods (such as, by way of example only, on a solid resin),by solution phase peptide synthesis methods, and/or without the aid ofenzymes, other embodiments of the non-natural amino acid polypeptidesdescribed herein allow synthesis via a cell membrane, cellular extract,or lysate system or via an in vivo system, such as, by way of exampleonly, using the cellular machinery of a prokaryotic or eukaryotic cell.In further or additional embodiments, one of the key features of thenon-natural amino acid polypeptides described herein is that they may besynthesized utilizing ribosomes. In further or additional embodiments ofthe non-natural amino acid polypeptides described herein are, thenon-natural amino acid polypeptides may be synthesized by a combinationof the methods including, but not limited to, a combination of solidresins, without the aid of enzymes, via the aid of ribosomes, and/or viaan in vivo system.

Synthesis of non-natural amino acid polypeptides via ribosomes and/or anin vivo system has distinct advantages and characteristic from anon-natural amino acid polypeptide synthesized on a solid resin orwithout the aid of enzymes. These advantages or characteristics includedifferent impurity profiles: a system utilizing ribosomes and/or an invivo system will have impurities stemming from the biological systemutilized, including host cell proteins, membrane portions, and lipids,whereas the impurity profile from a system utilizing a solid resinand/or without the aid of enzymes may include organic solvents,protecting groups, resin materials, coupling reagents and otherchemicals used in the synthetic procedures. In addition, the isotopicpattern of the non-natural amino acid polypeptide synthesized via theuse of ribosomes and/or an in vivo system may mirror the isotopicpattern of the feedstock utilized for the cells; on the other hand, theisotopic pattern of the non-natural amino acid polypeptide synthesizedon a solid resin and/or without the aid of enzymes may mirror theisotopic pattern of the amino acids utilized in the synthesis. Further,the non-natural amino acid synthesized via the use of ribosomes and/oran in vivo system may be substantially free of the D-isomers of theamino acids and/or may be able to readily incorporate internal cysteineamino acids into the structure of the polypeptide, and/or may rarelyprovide internal amino acid deletion polypeptides. On the other hand, anon-natural amino acid polypeptide synthesized via a solid resin and/orwithout the use of enzymes may have a higher content of D-isomers of theamino acids and/or a lower content of internal cysteine amino acidsand/or a higher percentage of internal amino acid deletion polypeptides.Furthermore, one of skill in the art will be able to differentiate anon-natural amino acid polypeptide synthesized by use of a ribosomeand/or an in vivo system from a non-natural amino acid polypeptidesynthesized via a solid resin and/or without the use of enzymes.

VII. Compositions and Methods Comprising Nucleic Acids andOligonucleotides

A. General Recombinant Nucleic Acid Methods for Use Herein

In numerous embodiments of the methods and compositions describedherein, nucleic acids encoding a polypeptide of interest (including byway of example a GH polypeptide) will be isolated, cloned and oftenaltered using recombinant methods. Such embodiments are used, includingbut not limited to, for protein expression or during the generation ofvariants, derivatives, expression cassettes, or other sequences derivedfrom a polypeptide. In some embodiments, the sequences encoding thepolypeptides are operably linked to a heterologous promoter.

A nucleotide sequence encoding a polypeptide comprising a non-naturalamino acid may be synthesized on the basis of the amino acid sequence ofthe parent polypeptide, and then changing the nucleotide sequence so asto effect introduction (i.e., incorporation or substitution) or removal(i.e., deletion or substitution) of the relevant amino acid residue(s).The nucleotide sequence may be conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence may be prepared by chemical synthesis, including butnot limited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction. See, e.g., Barany, et al.,Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 whichare incorporated by reference herein.

The non-natural amino acid methods and compositions described hereinutilize routine techniques in the field of recombinant genetics. Basictexts disclosing the general methods of use for the non-natural aminoacid methods and compositions described herein include Sambrook et al.,Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, GeneTransfer and Expression: A Laboratory Manual (1990); and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides whichinclude selector codons for production of proteins that includenon-natural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the non-natural amino acidmethods and compositions described herein for a variety of purposes,including but not limited to, to produce novel synthetases or tRNAs, tomutate tRNA molecules, to mutate polynucleotides encoding synthetases,libraries of tRNAs, to produce libraries of synthetases, to produceselector codons, to insert selector codons that encode non-natural aminoacids in a protein or polypeptide of interest. They include but are notlimited to site-directed mutagenesis, random point mutagenesis,homologous recombination, DNA shuffling or other recursive mutagenesismethods, chimeric construction, mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, or any combination thereof. Additional suitablemethods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the non-natural amino acid methods and compositionsdescribed herein. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, including but not limited to, sequencecomparisons, physical properties, crystal structure or the like.

The texts and examples found herein describe these and other relevantprocedures. Additional information is found in the followingpublications and references cited within: Ling et al., Approaches to DNAmutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997); Dale etal., Oligonucleotide-directed random mutagenesis using thephosphorothioate method, Methods Mol. Biol. 57:369-374 (1996); Smith, Invitro mutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein &Shortle, Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol.154: 329-350 (1987); Zoller & Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment,Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Taylor et al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8785(1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698(1986); Sayers et al., 5′-3′ Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802(1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Point Mismatch Repair, Cell 38:879-887(1984); Carter et al., Improved oligonucleotide site-directedmutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985);Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors,Methods in Enzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Useof oligonucleotides to generate large deletions, Nucl. Acids Res. 14:5115 (1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001). W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many suchmethods can be found in Methods in Enzymology Volume 154, which alsodescribes useful controls for trouble-shooting problems with variousmutagenesis methods.

The methods and compositions described herein also include use ofeukaryotic host cells, non-eukaryotic host cells, and organisms for thein vivo incorporation of a non-natural amino acid via orthogonal tRNA/RSpairs. Host cells are genetically engineered (including but not limitedto, transformed, transduced or transfected) with the polynucleotidescorresponding to the polypeptides described herein or constructs whichinclude a polynucleotide corresponding to the polypeptides describedherein, including but not limited to, a vector corresponding to thepolypeptides described herein, which can be, for example, a cloningvector or an expression vector. For example, the coding regions for theorthogonal tRNA, the orthogonal tRNA synthetase, and the protein to bederivatized are operably linked to gene expression control elements thatare functional in the desired host cell. The vector can be, for example,in the form of a plasmid, cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg N.Y.) and Atlas and Parks (eds) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in methods andcompositions described herein. These include: fusion of the recipientcells with bacterial protoplasts containing the DNA, electroporation,projectile bombardment, and infection with viral vectors (discussedfurther, herein), etc. Bacterial cells can be used to amplify the numberof plasmids containing DNA constructs corresponding to the polypeptidesdescribed herein. The bacteria are grown to log phase and the plasmidswithin the bacteria can be isolated by a variety of methods known in theart (see, for instance, Sambrook). In addition, a plethora of kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia BioteCH;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Gillam & Smith,Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, E.,et al., Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook,Berger (all supra). A catalogue of bacteria and bacteriophages usefulfor cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue ofbacteria and bacteriophage (1992) Gherna et al. (eds) published by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA Second EditionScientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex. mcrc.com), The Great American Gene Company (Ramona,Calif. available on the World Wide Web at genco.com), ExpressGen Inc.(Chicago, Ill. available on the World Wide Web at expressgen.com),Operon Technologies Inc. (Alameda, Calif.) and many others.

B. Selector Codons

Selector codons encompassed within the methods and compositionsdescribed herein expand the genetic codon framework of proteinbiosynthetic machinery. For example, a selector codon includes, but isnot limited to, a unique three base codon, a nonsense codon, such as astop codon, including but not limited to, an amber codon (UAG), or anopal codon (UGA), a unnatural codon, a four or more base codon, a rarecodon, or the like. There is a wide range in the number of selectorcodons that can be introduced into a desired gene or polynucleotide,including but not limited to, one or more, two or more, more than three,4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding atleast a portion of a polypeptide of interest.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more non-natural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O—RS with a desired non-natural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, UAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′,3′Exonuclease in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 16(3):791-802. When the O—RS, O-tRNA andthe nucleic acid that encodes the polypeptide of interest are combinedin vivo, the non-natural amino acid is incorporated in response to theUAG codon to give a polypeptide containing the non-natural amino acid atthe specified position.

The incorporation of non-natural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the methods and compositions describedherein includes using extended codons based on frameshift suppression.Four or more base codons can insert, including but not limited to, oneor multiple non-natural amino acids into the same protein. For example,in the presence of mutated O-tRNAs, including but not limited to, aspecial frameshift suppressor tRNAs, with anticodon loops, for example,with at least 8-10 nt anticodon loops, the four or more base codon isread as single amino acid. In other embodiments, the anticodon loops candecode, including but not limited to, at least a four-base codon, atleast a five-base codon, or at least a six-base codon or more. Sincethere are 256 possible four-base codons, multiple non-natural aminoacids can be encoded in the same cell using a four or more base codon.See, Anderson et al., (2002) Exploring the Limits of Codon and AnticodonSize, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding theGenetic Code: Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate non-naturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939-7945; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34-40. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194-12195. In an in vivo study, Moore et al. examinedthe ability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195-205. In one embodiment, extended codonsbased on rare codons or nonsense codons can be used in the methods andcompositions described herein, which can reduce missense readthrough andframeshift suppression at other unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182, and see also, Wu, Y.,et. al. (2002) J. Am. Chem. Soc. 124: 14626-14630. Other relevantpublications are listed herein.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322-8322; and Piccirilli et al., (1990) Nature, 343:33-37; Kool,(2000) Curr. Opin. Chem. Biol., 4:602-608. These bases in generalmispair to some degree with natural bases and cannot be enzymaticallyreplicated. Kool and co-workers demonstrated that hydrophobic packinginteractions between bases can replace hydrogen bonding to drive theformation of base pair. See, Kool, (2000) Curr. Opin. Chem. Biol.,4:602-608; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl.,36(24): 2825-2828. In an effort to develop an unnatural base pairsatisfying all the above requirements, Schultz, Romesberg and co-workershave systematically synthesized and studied a series of unnaturalhydrophobic bases. A PICS:PICS self-pair is found to be more stable thannatural base pairs, and can be efficiently incorporated into DNA byKlenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g.,McMinn et al., (1999) J. Am. Chem. Soc., 121:11585-11586; and Ogawa etal., (2000) J. Am. Chem. Soc., 122:3274-3278. A 3MN:3MN self-pair can besynthesized by KF with efficiency and selectivity sufficient forbiological function. See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc.,122:8803-8804. However, both bases act as a chain terminator for furtherreplication. A mutant DNA polymerase has been recently evolved that canbe used to replicate the PICS self pair. In addition, a 7AI self paircan be replicated. See, e.g., Tae et al., (2001) J. Am. Chem. Soc.,123:7439-7440. A novel metallobase pair, Dipic:Py, has also beendeveloped, which forms a stable pair upon binding Cu(II). See, Meggerset al., (2000) J. Am. Chem. Soc., 122:10714-10715. Because extendedcodons and unnatural codons are intrinsically orthogonal to naturalcodons, the non-natural amino acid methods described herein can takeadvantage of this property to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anon-natural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions described herein isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinunder “Mutagenesis and Other Molecular Biology Techniques” to include,for example, one or more selector codons for the incorporation of anon-natural amino acid. For example, a nucleic acid for a protein ofinterest is mutagenized to include one or more selector codons,providing for the incorporation of the one or more non-natural aminoacids. The methods and compositions described herein include any suchvariant, including but not limited to, mutant versions of any protein,for example, including at least one non-natural amino acid. Similarly,the methods and compositions described herein include correspondingnucleic acids, i.e., any nucleic acid with one or more selector codonsthat encodes or allows for the in vivo incorporation of one or morenon-natural amino acid.

Nucleic acid molecules encoding a polypeptide of interest, including byway of example only, GH polypeptide may be readily mutated to introducea cysteine at any desired position of the polypeptide. Cysteine iswidely used to introduce reactive molecules, water soluble polymers,proteins, or a wide variety of other molecules, onto a protein ofinterest. Methods suitable for the incorporation of cysteine into adesired position of a polypeptide are well known in the art, such asthose described in U.S. Pat. No. 6,608,183, which is herein incorporatedby reference in its entirety, and standard mutagenesis techniques. Theuse of such cysteine-introducing and utilizing techniques can be used inconjunction with the non-natural amino acid introducing and utilizingtechniques described herein.

VIII. In Vivo Generation of Polypeptides Comprising Non-Natural AminoAcids

For convenience, the in vivo generation of polypeptides comprisingnon-natural amino acids described in this section have been describedgenerically and/or with specific examples. However, the in vivogeneration of polypeptides comprising non-natural amino acids describedin this section should not be limited to just the generic descriptionsor specific example provided in this section, but rather the in vivogeneration of polypeptides comprising non-natural amino acids describedin this section apply equally well to all compounds that fall within thescope of Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII, includingany sub-formulas or specific compounds that fall within the scope ofFormulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII that are described inthe specification, claims and figures herein.

The polypeptides described herein can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Patent Application Publications 2003/0082575 (Ser. No.10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference in their entirety herein. These methodsinvolve generating a translational machinery that functionsindependently of the synthetases and tRNAs endogenous to the translationsystem (and are therefore sometimes referred to as “orthogonal”). In oneembodiment the translation system comprises a polynucleotide encodingthe polypeptide; the polynucleotide can be mRNA that was transcribedfrom the corresponding DNA, or the mRNA may arise from an RNA viralvector; further the polynucleotide comprises a selector codoncorresponding to the predesignated site of incorporation for thenon-natural amino acid. The translation system further comprises a tRNAfor and also when appropriate comprising the non-natural amino acid,where the tRNA is specific to/specifically recognizes the aforementionedselector codon; in further embodiments, the non-natural amino acid isaminoacylated. The non-natural amino acids include those having thestructure of any one of Formulas I-XVIII, XXX-XXXIV(A&B), andXXXX-XXXXIII described herein. In further or additional embodiments, thetranslation system comprises an aminoacyl synthetase specific for thetRNA, and in other or further embodiments, the translation systemcomprises an orthogonal tRNA and an orthogonal aminoacyl tRNAsynthetase. In further or additional embodiments, the translation systemcomprises at least one of the following: a plasmid comprising theaforementioned polynucleotide (such as, by way of example only, in theform of DNA), genomic DNA comprising the aforementioned polynucleotide(such as, by way of example only, in the form of DNA), or genomic DNAinto which the aforementioned polynucleotide has been integrated (infurther embodiments, the integration is stable integration). In furtheror additional embodiments of the translation system, the selector codonis selected from the group consisting of an amber codon, ochre codon,opal codon, a unique codon, a rare codon, an unnatural codon, afive-base codon, and a four-base codon. In further or additionalembodiments of the translation system, the tRNA is a suppressor tRNA. Infurther or additional embodiments, the non-natural amino acidpolypeptide is synthesized by a ribosome.

In further or additional embodiments, the translation system comprisesan orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase(O—RS). Typically, the O—RS preferentially aminoacylates the O-tRNA withat least one non-natural amino acid in the translation system and theO-tRNA recognizes at least one selector codon that is not recognized byother tRNAs in the system. The translation system thus inserts thenon-natural amino acid into a polypeptide produced in the system, inresponse to an encoded selector codon, thereby “substituting” anon-natural amino acid into a position in the encoded polypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for in the methodsdescribed herein to produce the non-natural amino acid polypeptidesdescribed herein. For example, keto-specific O-tRNA/aminoacyl-tRNAsynthetases are described in Wang, L., et al., Proc. Natl. Acad. Sci.USA 100(1):56-61 (2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746(2003). Exemplary O—RS, or portions thereof, are encoded bypolynucleotide sequences and include amino acid sequences disclosed inU.S. Patent Application Publications 2003/0082575 and 2003/0108885, eachincorporated herein by reference in their entirety. Corresponding O-tRNAmolecules for use with the O—RSs are also described in U.S. PatentApplication Publications 2003/0082575 (Ser. No. 10/126,927) and2003/0108885 (Ser. No. 10/126,931) which are incorporated by referencein their entirety herein. In addition, Mehl et al. in J. Am. Chem. Soc.2003; 125:935-939 and Santoro et al. Nature Biotechnology 2002 October;20:1044-1048, which are incorporated by reference in their entiretyherein, discuss screening methods and aminoacyl tRNA synthetase and tRNAmolecules for the incorporation of p-aminophenylalanine intopolypeptides

Exemplary O-tRNA sequences suitable for use in the methods describedherein include, but are not limited to, nucleotide sequences SEQ ID NOs:1-3 as disclosed in U.S. Patent Application Publication 2003/0108885(Ser. No. 10/126,931) which is incorporated by reference herein. Otherexamples of O-tRNA/aminoacyl-tRNA synthetase pairs specific toparticular non-natural amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference in its entirety herein. O—RS and O-tRNA thatincorporate both keto- and azide-containing amino acids in S. cerevisiaeare described in Chin, J. W., et al., Science 301:964-967 (2003).

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-natural amino acid. While any codoncan be used, it is generally desirable to select a codon that is rarelyor never used in the cell in which the O-tRNA/aminoacyl-tRNA synthetaseis expressed. By way of example only, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the polynucleotide coding sequence using mutagenesis methods known inthe art (including but not limited to, site-specific mutagenesis,cassette mutagenesis, restriction selection mutagenesis, etc.).

Methods for generating components of the protein biosynthetic machinery,such as O—RSs, O-tRNAs, and orthogonal O-tRNA/O—RS pairs that can beused to incorporate a non-natural amino acid are described in Wang, L.,et al., Science 292: 498-500 (2001); Chin, J. W., et al., J. Am. Chem.Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42: 6735-6746(2003). Methods and compositions for the in vivo incorporation ofnon-natural amino acids are described in U.S. Patent ApplicationPublication 2003/0082575 (Ser. No. 10/126,927) which is incorporated byreference in its entirety herein. Methods for selecting an orthogonaltRNA-tRNA synthetase pair for use in vivo translation system of anorganism are also described in U.S. Patent Application Publications2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference in its entirety herein.In addition PCT Publication No. WO 04/035743 entitled “Site SpecificIncorporation of Keto Amino Acids into proteins, which is incorporatedby reference in its entirety, describes orthogonal RS and tRNA pairs forthe incorporation of keto amino acids. PCT Publication No. WO 04/094593entitled “Expanding the Eukaryotic Genetic Code,” which is incorporatedby reference herein in its entirety, describes orthogonal RS and tRNApairs for the incorporation of non-naturally encoded amino acids ineukaryotic host cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O—RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as, by way of example only, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like, or a eukaryotic organism; (b) selecting (and/or screening) thelibrary of RSs (optionally mutant RSs) for members that aminoacylate anorthogonal tRNA (O-tRNA) in the presence of a non-natural amino acid anda natural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-natural amino acid, thereby providing the at least one recombinantO—RS; wherein the at least one recombinant O—RS preferentiallyaminoacylates the O-tRNA with the non-natural amino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. By way of example only, the inactiveRS can be generated by mutating at least about 1, at least about 2, atleast about 3, at least about 4, at least about 5, at least about 6, orat least about 10 or more amino acids to different amino acids,including but not limited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. By way of example only, themutant RSs can be generated by site-specific mutations, randommutations, diversity generating recombination mutations, chimericconstructs, rational design and by other methods described herein orknown in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RS's) for members that are active, including but notlimited to, those which aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-natural amino acid and a natural amino acid, includes,but is not limited to: introducing a positive selection or screeningmarker, including but not limited to, an antibiotic resistance gene, orthe like, and the library of (optionally mutant) RS's into a pluralityof cells, wherein the positive selection and/or screening markercomprises at least one selector codon, including but not limited to, anamber codon, ochre codon, opal codon, a unique codon, a rare codon, anunnatural codon, a five-base codon, and a four-base codon; growing theplurality of cells in the presence of a selection agent; identifyingcells that survive (or show a specific response) in the presence of theselection and/or screening agent by suppressing the at least oneselector codon in the positive selection or screening marker, therebyproviding a subset of positively selected cells that contains the poolof active (optionally mutant) RSs. Optionally, the selection and/orscreening agent concentration can be varied.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRS's (optionally mutants), including but not limited to, those whichpreferentially aminoacylate the O-tRNA in the absence of the non-naturalamino acid includes, but is not limited to: introducing a negativeselection or screening marker with the pool of active (optionallymutant) RS's from the positive selection or screening into a pluralityof cells of a second organism, wherein the negative selection orscreening marker comprises at least one selector codon (including butnot limited to, an antibiotic resistance gene, including but not limitedto, a chloramphenicol acetyltransferase (CAT) gene); and, identifyingcells that survive or show a specific screening response in a firstmedium supplemented with the non-natural amino acid and a screening orselection agent, but fail to survive or to show the specific response ina second medium not supplemented with the non-natural amino acid and theselection or screening agent, thereby providing surviving cells orscreened cells with the at least one recombinant O—RS. By way of exampleonly, a CAT identification protocol optionally acts as a positiveselection and/or a negative screening in determination of appropriateO—RS recombinants. For instance, a pool of clones is optionallyreplicated on growth plates containing CAT (which comprises at least oneselector codon) either with or without one or more non-natural aminoacid. Colonies growing exclusively on the plates containing non-naturalamino acids are thus regarded as containing recombinant O—RS. In oneaspect, the concentration of the selection (and/or screening) agent isvaried. In some aspects the first and second organisms are different.Thus, the first and/or second organism optionally comprises: aprokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, ayeast, an archaebacterium, a eubacterium, a plant, an insect, a protist,etc. In other embodiments, the screening marker comprises a fluorescentor luminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RS'sincludes, but is not limited to: isolating the pool of active mutantRS's from the positive selection step (b); introducing a negativeselection or screening marker, wherein the negative selection orscreening marker comprises at least one selector codon (including butnot limited to, a toxic marker gene, including but not limited to, aribonuclease barnase gene, comprising at least one selector codon), andthe pool of active (optionally mutant) RS's into a plurality of cells ofa second organism; and identifying cells that survive or show a specificscreening response in a first medium not supplemented with thenon-natural amino acid, but fail to survive or show a specific screeningresponse in a second medium supplemented with the non-natural aminoacid, thereby providing surviving or screened cells with the at leastone recombinant O—RS, wherein the at least one recombinant O—RS isspecific for the non-natural amino acid. In one aspect, the at least oneselector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In another embodiment, the methods for producing at least onerecombinant orthogonal aminoacyl-tRNA synthetase (O—RS) may furthercomprise: (d) isolating the at least one recombinant O—RS; (e)generating a second set of O—RS (optionally mutated) derived from the atleast one recombinant O—RS; and, (f) repeating steps (b) and (c) until amutated O—RS is obtained that comprises an ability to preferentiallyaminoacylate the O-tRNA. Optionally, steps (d)-(f) are repeated,including but not limited to, at least about two times. In one aspect,the second set of mutated O—RS derived from at least one recombinantO—RS can be generated by mutagenesis, including but not limited to,random mutagenesis, site-specific mutagenesis, recombination or acombination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-natural amino acid or an analogue. In one embodiment, the mutatedsynthetase is displayed on a cell surface, on a phage display or thelike.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include,but are not limited to: (a) generating a library of mutant tRNAs derivedfrom at least one tRNA, including but not limited to, a suppressor tRNA,from a first organism; (b) selecting (including but not limited to,negatively selecting) or screening the library for (optionally mutant)tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from asecond organism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O—RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O—RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-natural amino acid, wherein thenon-natural amino acid is biosynthesized in vivo either naturally orthrough genetic manipulation. The non-natural amino acid is optionallyadded to a growth medium for at least the first or second organism,wherein the non-natural amino acid is capable of achieving appropriateintracellular concentrations to allow incorporation into the non-naturalamino acid polypeptide.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods described herein,the toxic marker gene is a ribonuclease barnase gene, where theribonuclease barnase gene comprises at least one amber codon.Optionally, the ribonuclease barnase gene can include two or more ambercodons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS(O—RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O—RS , and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O—RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O—RS pairs are provided. Methodsinclude, but are not limited to: (a) generating a library of mutanttRNAs derived from at least one tRNA from a first organism; (b)negatively selecting or screening the library for (optionally mutant)tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from asecond organism in the absence of a RS from the first organism, therebyproviding a pool of (optionally mutant) tRNAs; (c) selecting orscreening the pool of (optionally mutant) tRNAs for members that areaminoacylated by an introduced orthogonal RS (O—RS), thereby providingat least one recombinant O-tRNA. The at least one recombinant O-tRNArecognizes a selector codon and is not efficiently recognized by the RSfrom the second organism and is preferentially aminoacylated by theO—RS. The method also includes (d) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a third organism; (e) selecting or screening the library of mutantRS's for members that preferentially aminoacylate the at least onerecombinant O-tRNA in the presence of a non-natural amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and, (f) negatively selecting or screening the pool foractive (optionally mutant) RSs that preferentially aminoacylate the atleast one recombinant O-tRNA in the absence of the non-natural aminoacid, thereby providing the at least one specific O-tRNA/O—RS pair,wherein the at least one specific O-tRNA/O—RS pair comprises at leastone recombinant O—RS that is specific for the non-natural amino acid andthe at least one recombinant O-tRNA. Specific O-tRNA/O—RS pairs producedby the methods described herein are included within the scope andmethods described herein. For example, the specific O-tRNA/O—RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe methods described herein. The methods include, but are not limitedto: introducing a marker gene, a tRNA and an aminoacyl-tRNA synthetase(RS) isolated or derived from a first organism into a first set of cellsfrom the second organism; introducing the marker gene and the tRNA intoa duplicate cell set from a second organism; and, selecting forsurviving cells in the first set that fail to survive in the duplicatecell set or screening for cells showing a specific screening responsethat fail to give such response in the duplicate cell set, wherein thefirst set and the duplicate cell set are grown in the presence of aselection or screening agent, wherein the surviving or screened cellscomprise the orthogonal tRNA-tRNA synthetase pair for use in the in thein vivo translation system of the second organism. In one embodiment,comparing and selecting or screening includes an in vivo complementationassay. The concentration of the selection or screening agent can bevaried.

The organisms described herein comprise a variety of organism and avariety of combinations. In one embodiment, the organisms are optionallya prokaryotic organism, including but not limited to, Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Halobacterium,Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the organisms are a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like.

A. Expression in Non-Eukaryotes and Eukaryotes

The techniques disclosed in this section can be applied to theexpression in non-eukaryotes and eukaryotes of the non-natural aminoacid polypeptides described herein.

To obtain high level expression of a cloned polynucleotide, onetypically subclones polynucleotides encoding a desired polypeptide intoan expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are described,e.g., in Sambrook et al. and Ausubel et al.

Bacterial expression systems for expressing polypeptides are availablein, including but not limited to, E. coli, Bacillus sp., Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida, and Salmonella(Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature302:543-545 (1983). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are commercially available. In cases where orthogonal tRNAsand aminoacyl tRNA synthetases (described elsewhere herein) are used toexpress the polypeptides, host cells for expression are selected basedon their ability to use the orthogonal components. Exemplary host cellsinclude Gram-positive bacteria (including but not limited to B. brevisor B. subtilis, or Streptomyces) and Gram-negative bacteria (E. coli orPseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), aswell as yeast and other eukaryotic cells. Cells comprising O-tRNA/O—RSpairs can be used as described herein.

A eukaryotic host cell or non-eukaryotic host cell as described hereinprovides the ability to synthesize polypeptides which comprisenon-natural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, but is not limited to, at least 10micrograms, at least 50 micrograms, at least 75 micrograms, at least 100micrograms, at least 200 micrograms, at least 250 micrograms, at least500 micrograms, at least 1 milligram, at least 10 milligrams, at least100 milligrams, at least one gram, or more of the polypeptide thatcomprises a non-natural amino acid, or an amount that can be achievedwith in vivo polypeptide production methods (details on recombinantprotein production and purification are provided herein). In anotheraspect, the polypeptide is optionally present in the composition at aconcentration of, including but not limited to, at least 10 microgramsof polypeptide per liter, at least 50 micrograms of polypeptide perliter, at least 75 micrograms of polypeptide per liter, at least 100micrograms of polypeptide per liter, at least 200 micrograms ofpolypeptide per liter, at least 250 micrograms of polypeptide per liter,at least 500 micrograms of polypeptide per liter, at least 1 milligramof polypeptide per liter, or at least 10 milligrams of polypeptide perliter or more, in, including but not limited to, a cell lysate, abuffer, a pharmaceutical buffer, or other liquid suspension (includingbut not limited to, in a volume of anywhere from about 1 nl to about 100L). The production of large quantities (including but not limited to,greater that that typically possible with other methods, including butnot limited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one non-natural amino acid is a feature of themethods, techniques and compositions described herein.

A eukaryotic host cell or non-eukaryotic host cell as described hereinprovides the ability to biosynthesize proteins that comprise non-naturalamino acids in large useful quantities. For example, polypeptidescomprising a non-natural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of polypeptide in acell extract, cell lysate, culture medium, a buffer, and/or the like.

1. Expression Systems, Culture, and Isolation

The techniques disclosed in this section can be applied to theexpression systems, culture and isolation of the non-natural amino acidpolypeptides described herein. Non-natural amino acid polypeptides maybe expressed in any number of suitable expression systems including, butnot limited to, yeast, insect cells, mammalian cells, and bacteria. Adescription of exemplary expression systems is provided herein.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding the non-natural amino acidpolypeptide. Such yeasts include, but are not limited to,ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts andyeasts belonging to the Fungi imperfecti (Blastomycetes) group. Theascosporogenous yeasts are divided into two families, Spermophthoraceaeand Saccharomycetaceae. The latter is comprised of four subfamilies,Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g., genera Pichia,Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts includethe genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium,and Filobasidiella. Yeasts belonging to the Fungi Imperfecti(Blastomycetes) group are divided into two families, Sporobolomycetaceae(e.g., genera Sporobolomyces and Bul/era) and Cryptococcaceae (e.g.,genus Candida).

In certain embodiments, the species within the genera Pichia,Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hansenula,Torulopsis, and Candida, including, but not limited to, P. pastoris, P.guillerimondii, S. cerevisiae, S. carlsbergensis, S. diastaticus, S.douglasii, S. kluyveri, S. norbensis, S. oviformis, K. lactis, K.fragilis, C. albicans, C. maltosa, and H. polymorpha are used in themethods, techniques and compositions described herein.

The selection of suitable yeast for expression of the non-natural aminoacid polypeptide is within the skill of one of ordinary skill in theart. In selecting yeast hosts for expression, suitable hosts mayinclude, but are not limited to, those shown to have, by way of example,good secretion capacity, low proteolytic activity, and overallrobustness. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a non-natural amino acidpolypeptide, are included in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1998) 112:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GEN. GENET. (1986) 202:302); Kfragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIO/TECHNOLOGY (1990) 8:135); P. guillerimondii (Kunze et al., J. BASICMICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach and Nurse, NATURE (1981) 300:706); andY lipolytica (Davidow et al., CURR. GENET. (1985) 10:380 (1985);Gaillardin et al., CURR. GENET. (1985) 10:49); A. nidulans (Ballance etal., BIOCHEM. BIOPHYS. RES. COMMUN. (1983) 112:284-89; Tilburn et al.,GENE (1983) 26:205-221; and Yelton et al., PROC. NATL. ACAD. SCI. USA(1984) 81:1470-74); A. niger (Kelly and Hynes, EMBO J. (1985)4:475-479); T reesia (EP 0 244 234); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357), each hereinincorporated by reference in their entirety

Control sequences for yeast vectors include, but are not limited to,promoter regions from genes such as alcohol dehydrogenase (ADH) (EP 0284 044); enolase; glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255(4):12073-12080); andother glycolytic enzymes, such as pyruvate decarboxylase,triosephosphate isomerase, and phosphoglucose isomerase (Holland et al.,BIOCHEMISTRY (1978) 17(23):4900-4907; Hess et al., J. ADV. ENZYME REG.(1969) 7:149-167). Inducible yeast promoters having the additionaladvantage of transcription controlled by growth conditions may includethe promoter regions for alcohol dehydrogenase 2; isocytochrome C; acidphosphatase; metallothionein; glyceraldehyde-3-phosphate dehydrogenase;degradative enzymes associated with nitrogen metabolism; and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP 0073657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. By way ofexample, the upstream activating sequences (UAS) of a yeast promoter maybe joined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein in their entirety.Other examples of hybrid promoters include promoters that consist of theregulatory sequences of the ADH2, GAL4, GAL10, or PHO5 genes, combinedwith the transcriptional activation region of a glycolytic enzyme genesuch as GAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter mayinclude naturally occurring promoters of non-yeast origin that have theability to bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts include, but arenot limited to, either the transformation of spheroplasts or of intactyeast host cells treated with alkali cations. By way of example,transformation of yeast can be carried out according to the methoddescribed in Hsiao et al., PROC. NATL. ACAD. SCI. USA (1979) 76:3829 andVan Solingen et al., J. BACT. (1977) 130:946. However, other methods forintroducing DNA into cells such as by nuclear injection,electroporation, or protoplast fusion may also be used as describedgenerally in SAMBROOK ET AL., MOLECULAR CLONING: A LAB. MANUAL (2001).Yeast host cells may then be cultured using standard techniques known tothose of ordinary skill in the art.

Other methods for expressing heterologous proteins in yeast host cellsare described in U.S. Patent Publication No. 20020055169, U.S. Pat. Nos.6,361,969; 6,312,923; 6,183,985; 6,083,723; 6,017,731; 5,674,706;5,629,203; 5,602,034; and 5,089,398; U.S. Reexamined Patent Nos.RE37,343 and RE35,749; PCT Published Patent Applications WO 99/07862; WO98/37208; and WO 98/26080; European Patent Applications EP 0 946 736; EP0 732 403; EP 0 480 480; WO 90/10277; EP 0 460 071; EP 0 340 986; EP 0329 203; EP 0 324 274; and EP 0 164 556. See also Gellissen et al.,ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93; Romanos et al., YEAST(1992) 8(6):423-488; Goeddel, METHODS IN ENZYMOLOGY (1990) 185:3-7, eachincorporated by reference herein in its entirety.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods. Thefermentation methods may be adapted to account for differences in aparticular yeast host's carbon utilization pathway or mode of expressioncontrol. By way of example only, fermentation of a Saccharomyces yeasthost may require a single glucose feed, complex nitrogen source (e.g.,casein hydrolysates), and multiple vitamin supplementation, whereas, themethylotrophic yeast P. pastoris may require glycerol, methanol, andtrace mineral feeds, but only simple ammonium (nitrogen) salts foroptimal growth and expression. See, e.g., U.S. Pat. No. 5,324,639;Elliott et al., J. PROTEIN CHEM. (1990) 9:95; and Fieschko et al.,BIOTECH. BIOENG. (1987) 29:1113, each incorporated by reference hereinin its entirety.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. By way of example, agrowth limiting nutrient, typically carbon, may be added to thefermentor during the amplification phase to allow maximal growth. Inaddition, fermentation methods generally employ a fermentation mediumdesigned to contain adequate amounts of carbon, nitrogen, basal salts,phosphorus, and other minor nutrients (vitamins, trace minerals andsalts, etc.). Examples of fermentation media suitable for use withPichia are described in U.S. Pat. Nos. 5,324,639 and 5,231,178, eachincorporated by reference herein in its entirety.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a non-natural amino acidpolypeptide, are included in the progeny intended by this definition.

The selection of suitable insect cells for expression of a polypeptideis well known to those of ordinary skill in the art. Several insectspecies are well described in the art and are commercially availableincluding, but not limited to, Aedes aegypti, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni. In selectinginsect hosts for expression, suitable hosts may include, but are notlimited to, those shown to have, inter alia, good secretion capacity,low proteolytic activity, and overall robustness. Insect are generallyavailable from a variety of sources including, but not limited to, theInsect Genetic Stock Center, Department of Biophysics and MedicalPhysics, University of California (Berkeley, Calif.); and the AmericanType Culture Collection (“ATCC”) (Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with a sequence homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). Illustrative techniquesare described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENTSTATION BULLETIN No. 1555 (1987), herein incorporated by reference. Seealso, RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUSEXPRESSION PROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS INMOLECULAR BIOLOGY 16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUSSYSTEM: A LABORATORY GUIDE (1992); and O'REILLY ET AL., BACULOVIRUSEXPRESSION VECTORS: A LABORATORY MANUAL (1992).

The production of various heterologous proteins using baculovirus/insectcell expression systems is described in the following references andsuch techniques can be adapted to produce the non-natural amino acidpolypeptides described herein. See, e.g., U.S. Pat. Nos. 6,368,825;6,342,216; 6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987;6,168,932; 6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285;5,891,676; 5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939;5,753,220; 5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686;WO02/06305; WO01/90390; WO01/27301; WO01/05956; WO00/55345; WO00/20032WO99/51721; WO99/45130; WO99/31257; WO99/10515; WO99/09193; WO97/26332;WO96/29400; WO96/25496; WO96/06161; WO95/20672; WO93/03173; WO92/16619;WO92/03628; WO92/01801; WO90/14428; WO90/10078; WO90/02566; WO90/02186;WO90/01556; WO89/01038; WO89/01037; WO88/07082., each incorporated byreference herein in its entirety.

Vectors that are useful in baculovirus/insect cell expression systemsinclude, but are not limited to, insect expression and transfer vectorsderived from the baculovirus Autographacalifornica nuclear polyhedrosisvirus (AcNPV), which is a helper-independent, viral expression vector.Viral expression vectors derived from this system usually use the strongviral polyhedrin gene promoter to drive expression of heterologousgenes. See generally, Reilly ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller et al., ANN. REV. MICROBIOL. (1988) 42:177) and aprokaryotic ampicillin-resistance (amp) gene and origin of replicationfor selection and propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31-39 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5NV5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Illustrative methods for introducing heterologous DNA into the desiredsite in the baculovirus virus described in SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31-39. By way of example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. See Miller et al., BIOESSAYS (1989) 4:91.

Transfection may be accomplished by electroporation using methodsdescribed in TROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995);Mann and King, J. GEN. VIROL. (1989) 70:3501. Alternatively, liposomesmay be used to transfect the insect cells with the recombinantexpression vector and the baculovirus. See, e.g., Liebman et al.,BIOTECHNIQUES (1999) 26(1):36; Graves et al., BIOCHEMISTRY (1998)37:6050; Nomura et al., J. BIOL. CHEM. (1998) 273(22):13570; Schmidt etal., PROTEIN EXPRESSION AND PURIFICATION (1998) 12:323; Siffert et al.,NATURE GENETICS (1998) 18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORYHANDBOOK 145-154 (1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION(1997) 10:263; Dolphin et al., NATURE GENETICS (1997) 17:491; Kost etal., GENE (1997) 190:139; Jakobsson et al., J. BIOL. CHEM. (1996)271:22203; Rowles et al., J. BIOL. CHEM. (1996) 271(37):22376; Reverseyet al., J. BIOL. CHEM. (1996) 271(39):23607-10; Stanley et al., J. BIOL.CHEM. (1995) 270:4121; Sisk et al., J. VIROL. (1994) 68(2):766; and Penget al., BIOTECHNIQUES (1993) 14.2:274. Commercially available liposomesinclude, for example, Cellfectin® and Lipofectin® (Invitrogen, Corp.,Carlsbad, Calif.). In addition, calcium phosphate transfection may beused. See T ROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995);Kitts, NAR (1990) 18(19):5667; and Mann and King, J. GEN. VIROL. (1989)70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765.

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques such as those described in Miller et al.,BIOESSAYS (1989)4:91; SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENTSTATION BULLETIN No. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See WO89/046,699; Wright, NATURE (1986) 321:718; Carbonell et al., J. VIROL.(1985) 56:153; Smith et al., MOL. CELL. BIOL. (1983) 3:2156. Seegenerally, Fraser et al., IN VITRO CELL. DEV. BIOL. (1989) 25:225. Morespecifically, the cell lines used for baculovirus expression vectorsystems commonly include, but are not limited to, Sf9 (Spodopterafrugiperda) (ATCC No. CRL-1711), Sf21 (Spodoptera frugiperda)(Invitrogen Corp., Cat. No. 11497-013 (Carlsbad, Calif.)), Tri-368(Trichopulsia ni), and High-Five™ BTI-TN-5B1-4 (Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression.

Bacteria. Bacterial expression techniques are well known in the art. Awide variety of vectors are available for use in bacterial hosts. Thevectors may be single copy or low or high multicopy vectors. Vectors mayserve for cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers are present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3″) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) (Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; IFNPub. Nos. 036776 and 121 775), each is herein incorporated by reference in itsentirety. The β-galactosidase (bla) promoter system [Weissmann (1981)“The cloning of interferon and other mistakes.” In Interferon 3 (Ed. I.Gresser)], bacteriophage lambda PL [Shimatake et al., NATURE (1981)292:128] and T5 [U.S. Pat. No. 4,689,406], each is herein incorporatedby reference in its entirety, promoter systems also provide usefulpromoter sequences. Preferred methods encompassed herein utilize strongpromoters, such as the T7 promoter to induce polypeptide production athigh levels. Examples of such vectors include, but are not limited to,the pET29 series from Novagen, and the pPOP vectors described inWO99/05297, which is herein incorporated by reference in its entirety.Such expression systems produce high levels of polypeptide in the hostwithout compromising host cell viability or growth parameters.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor [Amann et al., GENE (1983) 25:167; de Boer et al., PROC. NATL.ACAD. SCI. (1983) 80:21]. Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can also be coupledwith a compatible RNA polymerase to produce high levels of expression ofsome genes in prokaryotes. The bacteriophase T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (IFNPub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a desired polypeptide, are included in theprogeny intended by this definition.

The selection of suitable host bacteria for expression of a desiredpolypeptide is well known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may include,but are not limited to, those shown to have at least one of thefollowing characteristics, and preferably at least two of the followingcharacteristics, inter alia, good inclusion body formation capacity, lowproteolytic activity, good secretion capacity, good soluble proteinproduction capability, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. In one embodiment of the methods describedand encompassed herein, the E. coli host includes, but is not limitedto, strains of BL21, DH10B, or derivatives thereof. In anotherembodiment of the methods described and encompassed herein, the E. colihost is a protease minus strain including, but not limited to, OMP— andLON—. In another embodiment, the bacterial host is a species ofPseudomonas, such a P. fluorescens, P. aeruginosa, and P. putida. Anexample of a Pseudomonas expression strain is P. fluorescens biovar I,strain MB 101 (Dow Chemical).

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of polypeptides. The method of culture of the recombinanthost cell strain will be dependent on the nature of the expressionconstruct utilized and the identity of the host cell. Recombinant hoststrains are normally cultured using methods that are well known to theart. Recombinant host cells are typically cultured in liquid mediumcontaining assimilatable sources of carbon, nitrogen, and inorganicsalts and, optionally, containing vitamins, amino acids, growth factors,and other proteinaceous culture supplements well known to the art.Liquid media for culture of host cells may optionally containantibiotics or anti-fungals to prevent the growth of undesirablemicroorganisms and/or compounds including, but not limited to,antibiotics to select for host cells containing the expression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the desired polypeptideaccumulates intracellularly) or harvesting of culture supernatant ineither batch or continuous formats. For production in prokaryotic hostcells, batch culture and cell harvest are preferred.

In one embodiment, the non-natural amino acid polypeptides describedherein are purified after expression in recombinant systems. Thepolypeptides may be purified from host cells or culture medium by avariety of methods known to the art. Normally, many polypeptidesproduced in bacterial host cells are poorly soluble or insoluble (in theform of inclusion bodies). In one embodiment, amino acid substitutionsmay readily be made in the polypeptides that are selected for thepurpose of increasing the solubility of the recombinantly producedpolypeptide utilizing the methods disclosed herein, as well as thoseknown in the art. In the case of insoluble polypeptides, thepolypeptides may be collected from host cell lysates by centrifugationor filtering and may further be followed by homogenization of the cells.In the case of poorly soluble polypeptides, compounds including, but notlimited to, polyethylene imine (PEI) may be added to induce theprecipitation of partially soluble polypeptides. The precipitatedpolypeptides may then be conveniently collected by centrifugation orfiltering. Recombinant host cells may be disrupted or homogenized torelease the inclusion bodies from within the cells using a variety ofmethods well known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the methods described and encompassed herein, thehigh pressure release technique is used to disrupt the E. Coli hostcells to release the inclusion bodies of the polypeptides. It has beenfound that yields of insoluble polypeptides in the form of inclusionbodies may be increased by utilizing only one passage of the E. Colihost cells through the homogenizer. When handling inclusion bodies ofpolypeptides, it is advantageous to minimize the homogenization time onrepetitions in order to maximize the yield of inclusion bodies withoutloss due to factors such as solubilization, mechanical shearing orproteolysis.

Insoluble or precipitated polypeptides may then be solubilized using anyof a number of suitable solubilization agents known to the art. By wayof example, the polypeptides are solubilized with urea or guanidinehydrochloride. The volume of the solubilized polypeptides should beminimized so that large batches may be produced using convenientlymanageable batch sizes. This factor may be significant in a large-scalecommercial setting where the recombinant host may be grown in batchesthat are thousands of liters in volume. In addition, when manufacturingpolypeptides in a large-scale commercial setting, in particular forhuman pharmaceutical uses, the avoidance of harsh chemicals that candamage the machinery and container, or the polypeptide product itself,should be avoided, if possible. It has been shown in the methodsdescribed and encompassed herein that the milder denaturing agent ureacan be used to solubilize the polypeptide inclusion bodies in place ofthe harsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of a polypeptidewhile efficiently solubilizing the polypeptide inclusion bodies.

In the case of soluble polypeptides, the peptides may be secreted intothe periplasmic space or into the culture medium. In addition, solublepeptides may be present in the cytoplasm of the host cells. The solublepeptide may be concentrated prior to performing purification steps.Standard techniques, including but not limited to those describedherein, may be used to concentrate soluble peptide from, by way ofexample, cell lysates or culture medium. In addition, standardtechniques, including but not limited to those described herein, may beused to disrupt host cells and release soluble peptide from thecytoplasm or periplasmic space of the host cells.

When the polypeptide is produced as a fusion protein, the fusionsequence is preferably removed. Removal of a fusion sequence may beaccomplished by methods including, but not limited to, enzymatic orchemical cleavage, wherein enzymatic cleavage is preferred. Enzymaticremoval of fusion sequences may be accomplished using methods well knownto those in the art. The choice of enzyme for removal of the fusionsequence will be determined by the identity of the fusion, and thereaction conditions will be specified by the choice of enzyme. Chemicalcleavage may be accomplished using reagents, including but not limitedto, cyanogen bromide, TEV protease, and other reagents. The cleavedpolypeptide is optionally purified from the cleaved fusion sequence bywell known methods. Such methods will be determined by the identity andproperties of the fusion sequence and the polypeptide. Methods forpurification may include, but are not limited to, size-exclusionchromatography, hydrophobic interaction chromatography, ion-exchangechromatography or dialysis or any combination thereof.

The polypeptide is also optionally purified to remove DNA from theprotein solution. DNA may be removed by any suitable method known to theart, including, but not limited to, precipitation or ion exchangechromatography. In one embodiment, DNA is removed by precipitation witha nucleic acid precipitating agent, such as, but not limited to,protamine sulfate. The polypeptide may be separated from theprecipitated DNA using standard well known methods including, but notlimited to, centrifugation or filtration. Removal of host nucleic acidmolecules is an important factor in a setting where the polypeptide isto be used to treat humans and the methods described herein reduce hostcell DNA to pharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation may also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human forms of the non-natural amino acid polypeptides described hereincan generally be recovered using methods standard in the art. Forexample, culture medium or cell lysate can be centrifuged or filtered toremove cellular debris. The supernatant may be concentrated or dilutedto a desired volume or diatiltered into a suitable buffer to conditionthe preparation for further purification. Further purification of thenon-natural amino acid polypeptides described herein include, but arenot limited to, separating deamidated and clipped forms of a polypeptidevariant from the corresponding intact form.

Any of the following exemplary procedures can be employed forpurification of a non-natural amino acid polypeptide described herein:affinity chromatography; anion- or cation-exchange chromatography(using, including but not limited to, DEAE SEPHAROSE); chromatography onsilica; reverse phase HPLC; gel filtration (using, including but notlimited to, SEPHADEX G-75); hydrophobic interaction chromatography;size-exclusion chromatography, metal-chelate chromatography;ultratiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), SDS-PAGE, extraction, or anycombination thereof.

Polypeptides encompassed within the methods and compositions describedherein, including but not limited to, polypeptides comprisingnon-natural amino acids, antibodies to polypeptides comprisingnon-natural amino acids, binding partners for polypeptides comprisingnon-natural amino acids, may be purified, either partially orsubstantially to homogeneity, according to standard procedures known toand used by those of skill in the art. Accordingly, polypeptidesdescribed herein may be recovered and purified by any of a number ofmethods well known in the art, including but not limited to, ammoniumsulfate or ethanol precipitation, acid or base extraction, columnchromatography, affinity column chromatography, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, hydroxylapatite chromatography, lectin chromatography,gel electrophoresis and any combination thereof. Protein refolding stepscan be used, as desired, in making correctly folded mature proteins.High performance liquid chromatography (HPLC), affinity chromatographyor other suitable methods can be employed in final purification stepswhere high purity is desired. In one embodiment, antibodies made againstnon-natural amino acids (or polypeptides comprising non-natural aminoacids) are used as purification reagents, including but not limited to,for affinity-based purification of polypeptides comprising one or morenon-natural amino acid(s). Once purified, partially or to homogeneity,as desired, the polypeptides are optionally used for a wide variety ofutilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, but not limited to, those set forth in R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982); Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY;Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harrisand Angal (1990) Protein Purification Applications: A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice 3rdEdition Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing polypeptides comprising at least onenon-natural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the polypeptides will be folded in their nativeconformations. However, in certain embodiments of the methods andcompositions described herein, after synthesis, expression and/orpurification, the polypeptides may possess a conformation different fromthe desired conformations of the relevant polypeptides. In one aspect ofthe methods and compositions described herein, the expressed protein isoptionally denatured and then renatured. This optional denaturation andrenaturation is accomplished utilizing methods known in the art,including but not limited to, by adding a chaperonin to the polypeptideof interest, and by solubilizing the polypeptides in a chaotropic agentincluding, but not limited to, guanidine HCl, and utilizing proteindisulfide isomerase.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. By way of example, such re-folding maybe accomplished with the addition guanidine, urea, DTT, DTE, and/or achaperonin to a translation product of interest. Methods of reducing,denaturing and renaturing proteins are well known to those of skill inthe art (see, the references above, and Debinski, et al. (1993) J. Biol.Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconiug. Chem., 4:581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).Debinski, et al., for example, describe the denaturation and reductionof inclusion body proteins in guanidine-DTE. The proteins can berefolded in a redox buffer containing, including but not limited to,oxidized glutathione and L-arginine. Refolding reagents can be flowed orotherwise moved into contact with the one or more polypeptide or otherexpression product, or vice-versa.

In the case of prokaryotic production of a non-natural amino acidpolypeptide, the polypeptide thus produced may be misfolded and thuslacks or has reduced biological activity. The bioactivity of the proteinmay be restored by “refolding”. In one embodiment, a misfoldedpolypeptide is refolded by solubilizing (where the polypeptide is alsoinsoluble), unfolding and reducing the polypeptide chain using, by wayof example, one or more chaotropic agents (including, but not limitedto, urea and/or guanidine) and a reducing agent capable of reducingdisulfide bonds (including, but not limited to, dithiothreitol, DTT or2-mercaptoethanol, 2-ME). At a moderate concentration of chaotrope, anoxidizing agent is then added (including, but not limited to, oxygen,cystine or cystamine), which allows the reformation of disulfide bonds.An unfolded or misfolded polypeptide may be refolded using standardmethods known in the art, such as those described in U.S. Pat. Nos.4,511,502, 4,511,503, and 4,512,922, each of which is hereinincorporated by reference in its entirety. The polypeptide may also becofolded with other proteins to form heterodimers or heteromultimers.After refolding or cofolding, the polypeptide is optionally furtherpurified.

Purification of non-natural amino acid polypeptides may be accomplishedusing a variety of techniques, including but not limited those describedherein, by way of example hydrophobic interaction chromatography, sizeexclusion chromatography, ion exchange chromatography, reverse-phasehigh performance liquid chromatography, affinity chromatography, and thelike or any combination thereof. Additional purification may alsoinclude a step of drying or precipitation of the purified protein.

After purification, the non-natural amino acid polypeptides may beexchanged into different buffers and/or concentrated by any of a varietyof methods known to the art, including, but not limited to,diafiltration and dialysis. hGH that is provided as a single purifiedprotein may be subject to aggregation and precipitation. In certainembodiments the purified non-natural amino acid polypeptides may be atleast 90% pure (as measured by reverse phase high performance liquidchromatography, RP-HPLC, or sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, SDS-PAGE). In certain other embodiments the purifiednon-natural amino acid polypeptides may be at least 95% pure, or atleast 98% pure, or at least 99% or greater purity. Regardless of theexact numerical value of the purity of the non-natural amino acidpolypeptides, the non-natural amino acid polypeptides is sufficientlypure for use as a pharmaceutical product or for further processing,including but not limited to, conjugation with a water soluble polymersuch as PEG.

In certain embodiments the non-natural amino acid polypeptides moleculesmay be used as therapeutic agents in the absence of other activeingredients or proteins (other than excipients, carriers, andstabilizers, serum albumin and the like), and in certain embodiments thenon-natural amino acid polypeptides molecules they may be complexed withanother polypeptide or a polymer.

2. Purification of Non-Natural Amino Acid Polypeptides

General Purification Methods The techniques disclosed in this sectioncan be applied to the general purification of the non-natural amino acidpolypeptides described herein.

Any one of a variety of isolation steps may be performed on the celllysate extract, culture medium, inclusion bodies, periplasmic space ofthe host cells, cytoplasm of the host cells, or other materialcomprising the desired polypeptide or on any polypeptide mixturesresulting from any isolation steps including, but not limited to,affinity chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel filtration chromatography, highperformance liquid chromatography (“HPLC”), reversed phase-HPLC(“RP-HPLC”), expanded bed adsorption, or any combination and/orrepetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUW, fluorescence, pH, and conductivity. Examples of detectors includeMonitor UV-1, UVICORD® S II, Monitor UV-M II, Monitor UV-900, MonitorUPC-900, Monitor pH/C-900, and Conductivity Monitor (AmershamBiosciences, Piscataway, N.J.). Indeed, entire systems are commerciallyavailable including the various AKTA® systems from Amersham Biosciences(Piscataway, N.J.).

In one embodiment of the methods and compositions described herein, forexample, the polypeptide may be reduced and denatured by firstdenaturing the resultant purified polypeptide in urea, followed bydilution into TRIS buffer containing a reducing agent (such as DTT) at asuitable pH. In another embodiment, the polypeptide is denatured in ureain a concentration range of between about 2 M to about 9 M, followed bydilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.The refolding mixture of this embodiment may then be incubated. In oneembodiment, the refolding mixture is incubated at room temperature forfour to twenty-four hours. The reduced and denatured polypeptide mixturemay then be further isolated or purified.

As stated herein, the pH of the first polypeptide mixture may beadjusted prior to performing any subsequent isolation steps. Inaddition, the first polypeptide mixture or any subsequent mixturethereof may be concentrated using techniques known in the art. Moreover,the elution buffer comprising the first polypeptide mixture or anysubsequent mixture thereof may be exchanged for a buffer suitable forthe next isolation step using techniques well known to those of ordinaryskill in the art.

Ion Exchange Chromatography The techniques disclosed in this section canbe applied to the ion-chromatography of the non-natural amino acidpolypeptides described herein.

In one embodiment, and as an optional, additional step, ion exchangechromatography may be performed on the first polypeptide mixture. Seegenerally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No.18-1114-21, Amersham Biosciences (Piscataway, N.J.)). Commerciallyavailable ion exchange columns include HITRAP®, HIPREP®, and HILOAD®Columns (Amersham Biosciences, Piscataway, N.J.). Such columns utilizestrong anion exchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE®High Performance, and Q SEPHAROSE® XL; strong cation exchangers such asSP SEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SPSEPHAROSE® XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow;and weak cation exchangers such as CM SEPHAROSE® Fast Flow (AmershamBiosciences, Piscataway, N.J.). Anion or cation exchange columnchromatography may be performed on the polypeptide at any stage of thepurification process to isolate substantially purified polypeptide. Thecation exchange chromatography step may be performed using any suitablecation exchange matrix. Cation exchange matrices include, but are notlimited to, fibrous, porous, non-porous, microgranular, beaded, orcross-linked cation exchange matrix materials. Such cation exchangematrix materials include, but are not limited to, cellulose, agarose,dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, orcomposites of any of the foregoing. Following adsorption of thepolypeptide to the cation exchanger matrix, substantially purifiedpolypeptide may be eluted by contacting the matrix with a buffer havinga sufficiently high pH or ionic strength to displace the polypeptidefrom the matrix. Suitable buffers for use in high pH elution ofsubstantially purified polypeptide include, but are not limited to,citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging inconcentration from at least about 5 mM to at least about 100 mM.

Reverse-Phase Chromatography The techniques disclosed in this sectioncan be applied to the reverse-phase chromatography of the non-naturalamino acid polypeptides described herein.

RP-HPLC may be performed to purify proteins following suitable protocolsthat are known to those of ordinary skill in the art. See, e.g., Pearsonet al., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J.CHROM. (1983) 268:112-119; Kunitani et al., J. CHROM. (1986)359:391-402. RP-HPLC may be performed on the polypeptide to isolatesubstantially purified polypeptide. In this regard, silica derivatizedresins with alkyl functionalities with a wide variety of lengths,including, but not limited to, at least about C₃ to at least about C₃₀,at least about C₃ to at least about C₂₀, or at least about C₃ to atleast about C₁₈, resins may be used. Alternatively, a polymeric resinmay be used. For example, TosoHaas Amberchrome CG1000sd resin may beused, which is a styrene polymer resin. Cyano or polymeric resins with awide variety of alkyl chain lengths may also be used. Furthermore, theRP-HPLC column may be washed with a solvent such as ethanol. A suitableelution buffer containing an ion pairing agent and an organic modifiersuch as methanol, isopropanol, tetrahydrofuran, acetonitrile or ethanol,may be used to elute the polypeptide from the RP-HPLC column. The mostcommonly used ion pairing agents include, but are not limited to, aceticacid, formic acid, perchloric acid, phosphoric acid, trifluoroaceticacid, heptafluorobutyric acid, triethylamine, tetramethylammonium,tetrabutylammonium, triethylammonium acetate. Elution may be performedusing one or more gradients or isocratic conditions, with gradientconditions preferred to reduce the separation time and to decrease peakwidth. Another method involves the use of two gradients with differentsolvent concentration ranges. Examples of suitable elution buffers foruse herein may include, but are not limited to, ammonium acetate andacetonitrile solutions.

Hydrophobic Interaction Chromatography Purification Techniques Thetechniques disclosed in this section can be applied to the hydrophobicinteraction chromatography purification of the non-natural amino acidpolypeptides described herein.

Hydrophobic interaction chromatography (HIC) may be performed on thepolypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHYHANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, AmershamBiosciences (Piscataway, N.J.) which is incorporated by referenceherein. Suitable HIC matrices may include, but are not limited to,alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.). Briefly, prior to loading, the HIC column may beequilibrated using standard buffers known to those of ordinary skill inthe art, such as an acetic acid/sodium chloride solution or HEPEScontaining ammonium sulfate. Ammonium sulfate may be used as the bufferfor loading the HIC column. After loading the polypeptide, the columnmay then washed using standard buffers and conditions to remove unwantedmaterials but retaining the polypeptide on the HIC column. Thepolypeptide may be eluted with about 3 to about 10 column volumes of astandard buffer, such as a HEPES buffer containing EDTA and lowerammonium sulfate concentration than the equilibrating buffer, or anacetic acid/sodium chloride buffer, among others. A decreasing linearsalt gradient using, for example, a gradient of potassium phosphate, mayalso be used to elute the polypeptide molecules. The eluent may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute polypeptide.

Other Purification Techniques The techniques disclosed in this sectioncan be applied to other purification techniques of the non-natural aminoacid polypeptides described herein.

Yet another isolation step using, for example, gel filtration (GELFILTRATION: PRINCIPLES AND METHODS (Cat. No. 18-1022-18, AmershamBiosciences, Piscataway, N.J.) which is herein incorporated by referencein its entirety), hydroxyapatite chromatography (suitable matricesinclude, but are not limited to, HA-Ultrogel, High Resolution(Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-Gel HTPHydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first polypeptide mixture or any subsequent mixturethereof, to remove any excess salts and to replace the buffer with asuitable buffer for the next isolation step or even formulation of thefinal drug product. The yield of polypeptide, including substantiallypurified polypeptide, may be monitored at each step described hereinusing various techniques, including but not limited those describedherein. Such techniques may also used to assess the yield ofsubstantially purified polypeptide following the last isolation step. Byway of example, the yield of polypeptide may be monitored using any ofseveral reverse phase high pressure liquid chromatography columns,having a variety of alkyl chain lengths such as cyano RP-HPLC,C₁₈RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring polypeptide using Western blot and ELISA assays. Forexample, polyclonal antibodies may be generated against proteinsisolated from negative control yeast fermentation and the cationexchange recovery. The antibodies may also be used to probe for thepresence of contaminating host cell proteins.

In certain embodiments, the yield of polypeptide after each purificationstep may be at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, at least about 99.9%, or at least about99.99%, of the polypeptide in the starting material for eachpurification step.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of polypeptidefrom the proteinaceous impurities is based on differences in thestrength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silica gel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoro-acetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The polypeptide fractions which arewithin the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of polypeptide to the DEAE groups is mediated byionic interactions. Acetonitrile and trifluoroacetic acid pass throughthe column without being retained. After these substances have beenwashed off, trace impurities are removed by washing the column withacetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and polypeptide is eluted with a buffer with increasedionic strength. The column is packed with DEAE Sepharose fast flow. Thecolumn volume is adjusted to assure a polypeptide load in the range of3-10 mg polypeptide/ml gel. The column is washed with water andequilibration buffer (sodium/potassium phosphate). The pooled fractionsof the HPLC eluate are loaded and the column is washed withequilibration buffer. Then the column is washed with washing buffer(sodium acetate buffer) followed by washing with equilibration buffer.Subsequently, polypeptide is eluted from the column with elution buffer(sodium chloride, sodium/potassium phosphate) and collected in a singlefraction in accordance with the master elution profile. The eluate ofthe DEAE Sepharose column is adjusted to the specified conductivity. Theresulting drug substance is sterile filtered into Teflon bottles andstored at −70° C.

A wide variety of methods and procedures can be used to assess the yieldand purity of a polypeptide one or more non-natural amino acids,including but not limited to, the Bradford assay, SDS-PAGE, silverstained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry(including but not limited to, MALDI-TOF) and other methods forcharacterizing proteins known to one skilled in the art.

Additional methods include, but are not limited to, steps to removeendotoxins. Endotoxins are lipopoly-saccharides (LPSs) which are locatedon the outer membrane of Gram-negative host cells, such as, for example,Escherichia coli. Methods for reducing endotoxin levels include, but arenot limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.

Additional methods and procedures include, but are not limited to,SDS-PAGE coupled with protein staining methods, immunoblotting, matrixassisted laser desorption/ionization-mass spectrometry (MALDI-MS),liquid chromatography/mass spectrometry, isoelectric focusing,analytical anion exchange, chromatofocusing, and circular dichroism

In certain embodiments amino acids of Formulas I-XVIII, XXX-XXXIV(A&B),and XXXX-XXXXIII, including any sub-formulas or specific compounds thatfall within the scope of Formulas I-XVIII, XXX-XXXIV(A&B), andXXXX-XXXXIII may be biosynthetically incorporated into polypeptides,thereby making non-natural amino acid polypeptides. In otherembodiments, such amino acids are incorporated at a specific site withinthe polypeptide. In other embodiments, such amino acids incorporatedinto the polypeptide using a translation system. In other embodiments,such translation systems comprise: (i) a polynucleotide encoding thepolypeptide, wherein the polynucleotide comprises a selector codoncorresponding to the pre-designated site of incorporation of the aboveamino acids, and (ii) a tRNA comprising the amino acid, wherein the tRNAis specific to the selector codon. In other embodiments of suchtranslation systems, the polynucleotide is mRNA produced in thetranslation system. In other embodiments of such translation systems,the translation system comprises a plasmid or a phage comprising thepolynucleotide. In other embodiments of such translation systems, thetranslation system comprises genomic DNA comprising the polynucleotide.In other embodiments of such translation systems, the polynucleotide isstably integrated into the genomic DNA. In other embodiments of suchtranslation systems, the translation system comprises tRNA specific fora selector codon selected from the group consisting of an amber codon,ochre codon, opal codon, a unique codon, a rare codon, an unnaturalcodon, a five-base codon, and a four-base codon. In other embodiments ofsuch translation systems, the tRNA is a suppressor tRNA. In otherembodiments of such translation systems, the translation systemcomprises a tRNA that is aminoacylated to the amino acids above. Inother embodiments of such translation systems, the translation systemcomprises an aminoacyl synthetase specific for the tRNA. In otherembodiments of such translation systems, the translation systemcomprises an orthogonal tRNA and an orthogonal aminoacyl tRNAsynthetase. In other embodiments of such translation systems, thepolypeptide is synthesized by a ribosome, and in further embodiments thetranslation system is an in vivo translation system comprising a cellselected from the group consisting of a bacterial cell, archeaebacterialcell, and eukaryotic cell. In other embodiments the cell is anEscherichia coli cell, yeast cell, a cell from a species of Pseudomonas,mammalian cell, plant cell, or an insect cell. In other embodiments ofsuch translation systems, the translation system is an in vitrotranslation system comprising cellular extract from a bacterial cell,archeaebacterial cell, or eukaryotic cell. In other embodiments, thecellular extract is from an Escherichia coli cell, a cell from a speciesof Pseudomonas, yeast cell, mammalian cell, plant cell, or an insectcell. In other embodiments at least a portion of the polypeptide issynthesized by solid phase or solution phase peptide synthesis, or acombination thereof, while in other embodiments further compriseligating the polypeptide to another polypeptide. In other embodimentsamino acids of Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII,including any sub-formulas or specific compounds that fall within thescope of Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII may bebiosynthetically incorporated into polypeptides, wherein the polypeptideis a protein homologous to a therapeutic protein selected from the groupconsisting of: alpha-1 antitrypsin, angiostatin, antihemolytic factor,antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrialnatriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2,ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

B. In Vivo Post-Translational Modifications

By producing polypeptides of interest with at least one non-naturalamino acid in eukaryotic cells, such polypeptides may include eukaryoticpost-translational modifications. In certain embodiments, a proteinincludes at least one non-natural amino acid and at least onepost-translational modification that is made in vivo by a eukaryoticcell, where the post-translational modification is not made by aprokaryotic cell. By way of example, the post-translation modificationincludes, but is not limited to, acetylation, acylation,lipid-modification, palmitoylation, palmitate addition, phosphorylation,glycolipid-linkage modification, glycosylation, and the like. In oneaspect, the post-translational modification includes attachment of anoligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 1 EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type BaseStructure High-mannose

Hybrid

Complex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of a non-natural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the non-natural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. Inpolypeptides described herein or produced using the methods describedherein, other more selective reactions can be used, including, but notlimited to, the reaction of a non-natural keto-amino acid withhydrazides or aminooxy compounds, in vitro and in vivo. See, e.g.,Cornish, et al., (1996) Am. Chem. Soc., 118:8150-8151; Mahal, et al.,(1997) Science, 276:1125-1128; Wang, et al., (2001) Science 292:498-500;Chin, et al., (2002) Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002)Proc. Natl. Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl.Acad. Sci., 100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746;and, Chin, et al., (2003) Science 300:964-967. This allows the selectivelabeling of virtually any protein with a host of reagents includingfluorophores, crosslinking agents, saccharide derivatives and cytotoxicmolecules. See also, U.S. patent application Ser. No. 10/686,944entitled “Glycoprotein synthesis” filed Jan. 16, 2003, which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligtation, PNAS 99(1): 19-24.

IX. Alternate Systems For Producing Non-Natural Amino Acid Polypeptides

Several strategies have been employed to introduce non-natural aminoacids into proteins in non-recombinant host cells, mutagenized hostcells, or in cell-free systems. The alternate systems disclosed in thissection can be applied to production of the non-natural amino acidpolypeptides described herein. By way of example, derivatization ofamino acids with reactive side-chains such as Lys, Cys and Tyr resultsin the conversion of lysine to N²-acetyl-lysine. Chemical synthesis alsoprovides a straightforward method to incorporate non-natural aminoacids. With the recent development of enzymatic ligation and nativechemical ligation of peptide fragments, it is possible to make largerproteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev.Biochem., 69:923 (2000). Chemical peptide ligation and native chemicalligation are described in U.S. Pat. No. 6,184,344, U.S. PatentPublication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046,WO 02/098902, and WO 03/042235, which are herein incorporated byreference in their entirety. A general in vitro biosynthetic method inwhich a suppressor tRNA chemically acylated with the desired non-naturalamino acid is added to an in vitro extract capable of supporting proteinbiosynthesis, has been used to site-specifically incorporate over 100non-natural amino acids into a variety of proteins of virtually anysize. See, e.g., V. W. Cornish, D. Mendel and P. G. Schultz, Angew.Chem. Int. Ed. Engl., 1995, 34:621-633 (1995); C. J. Noren, S. J.Anthony-Cahill, M. C. Griffith, P. G. Schultz, A general method forsite-specific incorporation of unnatural amino acids into proteins,Science 244 182-188 (1989); and, J. D. Bain, C. G. Glabe, T. A. Dix, A.R. Chamberlin, E. S. Diala, Biosynthetic site-specific incorporation ofa non-natural amino acid into a polypeptide, J. Am. Chem. Soc. 1118013-8014 (1989). A broad range of functional groups has been introducedinto proteins for studies of protein stability, protein folding, enzymemechanism, and signal transduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41-51 (1999). An auxotrophic strain,in which the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the non-natural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe non-natural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UW spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29-34 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404-3416 (1997);and trifluoroleucine has been incorporated in place of leucine,resulting in increased thermal and chemical stability of aleucine-zipper protein. See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka,T. Nakajima, W. F. DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed.Engl., 40(8):1494-1496 (2001). Moreover, selenomethionine andtelluromethionine are incorporated into various recombinant proteins tofacilitate the solution of phases in X-ray crystallography. See, e.g.,W. A. Hendrickson, J. R. Horton and D. M. Lemaster, EMBO J.,9(5):1665-1672 (1990); J. O. Boles, K. Lewinski, M. Kunkle, J. D. Odom,B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct. Biol., 1:283-284(1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J. Kellermannand R. Huber, Eur. J. Biochem., 230:788-796 (1995); and, N. Budisa, W.Kambrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind, L. Moroderand R. Huber, J. Mol. Biol., 270:616-623 (1997). Methionine analogs withalkene or alkyne functionalities have also been incorporatedefficiently, allowing for additional modification of proteins bychemical means. See, e.g., J. C. M. vanhest and D. A. Tirrell, FEBSLett, 428:68-70 (1998); J. C. M. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282-1288 (2000); and, K. L. Kiick andD. A. Tirrell, Tetrahedron, 56:9487-9493 (2000); U.S. Pat. No.6,586,207; U.S. Patent Publication 2002/0042097, which are hereinincorporated by reference in their entirety.

The success of this method depends on the recognition of the non-naturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. By way of example only, replacement of Ala²⁹⁴by Gly in Escherichia coli phenylalanyl-tRNA synthetase (PheRS)increases the size of substrate binding pocket, and results in theacylation of tRNAPhe by p-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P.Kast and H. Hennecke, Biochemistry, 33:7107-7112 (1994). An Escherichiacoli strain harboring this mutant PheRS allows the incorporation ofp-Cl-phenylalanine or p-Br-phenylalanine in place of phenylalanine. See,e.g., M. Ibba and H. Hennecke, FEBS Lett, 364:272-275 (1995); and, N.Sharma, R. Furter, P. Kast and D. A. Tirrell, FEBS Lett., 467:37-40(2000). Similarly, a point mutation Phe130Ser near the amino acidbinding site of Escherichia coli tyrosyl-tRNA synthetase was shown toallow azatyrosine to be incorporated more efficiently than tyrosine.See, F. Hamano-Takaku, T. Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M.Kitabatake, D. Soll and S, Nishimura, J. Biol. Chem.,275(51):40324-40328 (2000).

Another strategy to incorporate non-natural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501-504 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. J. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192(4809):1227-1232 (1961); Hofmann, K., Bohn, H. Studies onpolypeptides. XXXVI. The effect of pyrazole-imidazole replacements onthe S-protein activating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22(2):47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T.Peptide segment coupling catalyzed by the semisynthetic enzymethiosubtilisin, J Am Chem Soc, 109, 3808-3810 (1987); Schnolzer, M.,Kent, S B H. Constructing proteins by dovetailing unprotected syntheticpeptides: backbone-engineered HIV protease, Science, 256, 221-225(1992); Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit.Rev Biochem, 255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1 (3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266, 243-247 (1994).

Chemical modification has been used to introduce a variety ofnon-natural side chains, including cofactors, spin labels andoligonucleotides into proteins in vitro. See, e.g., Corey, D. R.,Schultz, P. G. Generation of a hybrid sequence-specific single-strandeddeoxyribonuclease, Science, 238, 1401-1403 (1987); Kaiser, E. T.,Lawrence D. S., Rokita, S. E. The chemical modification of enzymaticspecificity, Ann. Rev Biochem, 54, 565-595 (1985); Kaiser, E. T.,Lawrence, D. S. Chemical mutation of enyzme active sites, Science, 226,505-511 (1984); Neet, K. E., Nanci A, Koshland, D. E. Properties ofthiol-subtilisin, J. Biol. Chem., 243(24):6392-6401 (1968); Polgar, L.B., M. L. A new enzyme containing a synthetically formed active site.Thiol-subtilisin. J. Am. Chem Soc, 88(13):3153-3154 (1966); and,Pollack, S. J., Nakayama, G. Schultz, P. G. Introduction of nucleophilesand spectroscopic probes into antibody combining sites, Science,1(242):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 483-514 (1993); and, Krieg, U. C., Walter,P., Hohnson, A. E. Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognitionparticle, Proc. Natl. Acad. Sci, 83, 8604-8608 (1986).

Previously, it has been shown that non-natural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotrophic for a particular aminoacid. See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz,P. G. A general method for site-specific incorporation of unnaturalamino acids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, etal., Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., 202, 301-336 (1992);and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with a non-natural aminoacid. Conventional site-directed mutagenesis was used to introduce thestop codon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′,3′ Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the non-natural amino acid was incorporated in response to theUAG codon which gave a protein containing that amino acid at thespecified position. Experiments using [³H]-Phe and experiments withα-hydroxy acids demonstrated that only the desired amino acid isincorporated at the position specified by the UAG codon and that thisamino acid is not incorporated at any other site in the protein. See,e.g., Noren, et al, supra; Kobayashi et al., (2003) Nature StructuralBiology 10(6):425-432; and, Ellman, J. A., Mendel, D., Schultz, P. G.Site-specific incorporation of novel backbone structures into proteins,Science, 255, 197-200 (1992).

Microinjection techniques have also been used to incorporate non-naturalamino acids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439-442 (1995); and, D. A. Dougherty, Curr.Opin. Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected withtwo RNA species made in vitro: an mRNA encoding the target protein witha UAG stop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired non-natural amino acid.The translational machinery of the oocyte then inserts the non-naturalamino acid at the position specified by UAG. This method has allowed invivo structure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples include,but are not limited to, the incorporation of a fluorescent amino acidinto tachykinin neurokinin-2 receptor to measure distances byfluorescence resonance energy transfer, see, e.g., G. Turcatti, K.Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Knowles,H. Vogel and A. Chollet, J. Biol. Chem., 271(33): 19991-19998 (1996);the incorporation of biotinylated amino acids to identifysurface-exposed residues in ion channels, see, e.g., J. P. Gallivan, H.A. Lester and D. A. Dougherty, Chem. Biol., 4(10):739-749 (1997); theuse of caged tyrosine analogs to monitor conformantional changes in anion channel in real time, see, e.g., J. C. Miller, S. K. Silverman, P.M. England, D. A. Dougherty and H. A. Lester, Neuron, 20:619-624 (1998);and, the use of alpha hydroxy amino acids to change ion channelbackbones for probing their gating mechanisms. See, e.g., P. M. England,Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89-98 (1999); and,T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J. Yang,Nat. Neurosci., 4(3):239-246 (2001).

The ability to incorporate non-natural amino acids directly intoproteins in vivo offers the advantages of high yields of mutantproteins, technical ease, the potential to study the mutant proteins incells or possibly in living organisms and the use of these mutantproteins in therapeutic treatments. The ability to include non-naturalamino acids with various sizes, acidities, nucleophilicities,hydrophobicities, and other properties into proteins can greatly expandour ability to rationally and systematically manipulate the structuresof proteins, both to probe protein function and create new proteins ororganisms with novel properties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419-426 (1998).

It may also be possible to obtain expression of a desired polynucleotideusing a cell-free (in-vitro) translational system. In these systems,which can include either mRNA as a template (in-vitro translation) orDNA as a template (combined in-vitro transcription and translation), thein vitro synthesis is directed by the ribosomes. Considerable effort hasbeen applied to the development of cell-free protein expression systems.See, e.g., Kim, D.-M. and J. R. Swartz, Biotechnology andBioengineering, 74(4):309-316 (2001); Kim, D.-M. and J. R. Swartz,Biotechnology Letters, 22, 1537-1542, (2000); Kim, D.-M., and J. R.Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D.-M., and J.R. Swartz, Biotechnology and Bioengineering, 66(3): 180-188, (1999); andPatnaik, R. and J. R. Swartz, Biotechniques 24(5): 862-868, (1998); U.S.Pat. No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are herein incorporated by reference intheir entirety. Another approach that may be applied to the expressionof polypeptides comprising a non-natural amino acid includes, but is notlimited to, the mRNA-peptide fusion technique. See, e.g., R. Roberts andJ. Szostak, Proc. Natl. Acad. Sci. (USA) 94 12297-12302 (1997); A.Frankel, et al., Chemistry & Biology 10, 1043-1050 (2003). In thisapproach, an mRNA template linked to puromycin is translated intopeptide on the ribosome. If one or more tRNA molecules has beenmodified, non-natural amino acids can be incorporated into the peptideas well. After the last mRNA codon has been read, puromycin captures theC-terminus of the peptide. If the resulting mRNA-peptide conjugate isfound to have interesting properties in an in vitro assay, its identitycan be easily revealed from the mRNA sequence. In this way, one mayscreen libraries of polypeptides comprising one or more non-naturalamino acids to identify polypeptides having desired properties. Morerecently, in vitro ribosome translations with purified components havebeen reported that permit the synthesis of peptides substituted withnon-natural amino acids. See, e.g., A. Forster et al., Proc. Natl. Acad.Sci. (USA) 100(11): 6353-6357 (2003).

X. Post-Translational Modifications of Non-Natural Amino Acid Componentsof a Polypeptide

For convenience, the post-translational modifications of non-naturalamino acid components of a polypeptide described in this section (XA toXJ) have been described generically and/or with specific examples.However, the post-translational modifications of non-natural amino acidcomponents of a polypeptide described in this section should not belimited to just the generic descriptions or specific example provided inthis section, but rather the post-translational modifications ofnon-natural amino acid components of a polypeptide described in thissection apply equally well to all compounds that fall within the scopeof Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII, including anysub-formulas or specific compounds that fall within the scope ofFormulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII that are described inthe specification, claims and figures herein.

Methods, compositions, techniques and strategies have been developed tosite-specifically incorporate non-natural amino acids during the in vivotranslation of proteins. By incorporating a non-natural amino acid witha sidechain chemistry that is orthogonal to those of thenaturally-occurring amino acids, this technology makes possible thesite-specific derivatization of recombinant proteins. As a result, amajor advantage of the methods, compositions, techniques and strategiesdescribed herein is that derivatized proteins can now be prepared asdefined homogeneous products. However, the methods, compositions,reaction mixtures, techniques and strategies described herein are notlimited to non-natural amino acid polypeptides formed by in vivo proteintranslation techniques, but includes non-natural amino acid polypeptidesformed by any technique, including by way of example only expressedprotein ligation, chemical synthesis, ribozyme-based techniques (see,e.g., section herein entitled “Expression in Alternate Systems”).

The ability to incorporate non-natural amino acids into recombinantproteins broadly expands the chemistries which may be implemented forpost-translational derivatization, wherein such derivatization occurseither in vivo or in vitro. More specifically, protein derivatization toform an oxime linkage on a non-natural amino acid portion of apolypeptide offers several advantages. First, the naturally occurringamino acids generally do not form oxime linkages and thus reagentsdesigned to form oxime linkages will react site-specifically with thenon-natural amino acid component of the polypeptide (assuming of coursethat the non-natural amino acid and the corresponding reagent have beendesigned to form an oxime linkage), thus the ability to site-selectivelyderivatize proteins provides a single homogeneous product as opposed tothe mixtures of derivatized proteins produced using prior arttechnology. Second, oxime adducts are stable under biologicalconditions, suggesting that proteins derivatized by oxime exchange arevalid candidates for therapeutic applications. Third, the stability ofthe resulting oxime linkage can be manipulated based on the identity(i.e., the functional groups and/or structure) of the non-natural aminoacid to which the oxime linkage has been formed. Thus, as shown inExample 16, the pH stability of the oxime linkage to a non-natural aminoacid can vary from less than an hour to significantly more than a week.Thus, in some embodiments, the oxime linkage to the non-natural aminoacid polypeptide has a decomposition half life less than one hour, inother embodiments less than 1 day, in other embodiments less than 2days, in other embodiments less than 1 week and in other embodimentsmore than 1 week. In yet other embodiments, the resulting oxime isstable for at least two weeks under mildly acidic conditions, in otherembodiments the resulting oxime is stable for at least 5 days undermildly acidic conditions. In other embodiments, the non-natural aminoacid polypeptide is stable for at least 1 day in a pH between about 2and about 8; in other embodiments, from a pH of about 2 to about 6; inother embodiment, in a pH of about 2 to about 4. In other embodiments,using the strategies, methods, compositions and techniques describedherein, one of skill in the art will be able to synthesize an oximelinkage to a non-natural amino acid polypeptide with a decompositionhalf-life tuned to the needs of that skilled artisan (e.g., for atherapeutic use such as sustained release, or a diagnostic use, or anindustrial use or a military use).

The non-natural amino acid polypeptides described above are useful for,including but not limited to, novel therapeutics, diagnostics, catalyticenzymes, industrial enzymes, binding proteins (including but not limitedto, antibodies and antibody fragments), and including but not limitedto, the study of protein structure and function. See, e.g., Dougherty,(2000) Unnatural Amino Acids as Probes of Protein Structure andFunction, Current Opinion in Chemical Biology, 4:645-652. Other uses forthe non-natural amino acid polypeptides described above include, by wayof example only, assay-based, cosmetic, plant biology, environmental,energy-production, and/or military uses. However, the non-natural aminoacid polypeptides described above can undergo further modifications soas to incorporate new or modified functionalities, includingmanipulating the therapeutic effectiveness of the polypeptide, improvingthe safety profile of the polypeptide, adjusting the pharmacokinetics,pharmacologics and/or pharmacodynamics of the polypeptide (e.g.,increasing water solubility, bioavailability, increasing serumhalf-life, increasing therapeutic half-life, modulating immunogenicity,modulating biological activity, or extending the circulation time),providing additional functionality to the polypeptide, incorporating atag, label or detectable signal into the polypeptide, easing theisolation properties of the polypeptide, and any combination of theaforementioned modifications.

In certain embodiments are methods for easing the isolation propertiesof a polypeptide comprising utilizing a homologous biosyntheticnon-natural amino acid polypeptide comprising at least one non-naturalamino acid selected from the group consisting of an oxime-containingnon-natural amino acid, a carbonyl-containing non-natural amino acid,and a hydroxylamine-containing non-natural amino acid. In otherembodiments such non-natural amino acids have been biosyntheticallyincorporated into the polypeptide as described herein. In further oralternative embodiments such non-natural amino acid polypeptidescomprise at least one non-natural amino acid selected from amino acidsof Formula I-XVIII, XXX-XXXIV(A&B), or XXXX-XXXXIII.

The methods, compositions, strategies and techniques described hereinare not limited to a particular type, class or family of polypeptides.Indeed, virtually any polypeptide may include at least one non-naturalamino acids described herein. By way of example only, the polypeptidecan be homologous to a therapeutic protein selected from the groupconsisting of: alpha-1 antitrypsin, angiostatin, antihemolytic factor,antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrialnatriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2,ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone. The non-natural amino acid polypeptide may also behomologous to any polypeptide member of the growth hormone supergenefamily.

Such modifications include the incorporation of further functionalityonto the non-natural amino acid component of the polypeptide, includingbut not limited to, a label; a dye; a polymer; a water-soluble polymer;a derivative of polyethylene glycol; a photocrosslinker; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide, a water-soluble dendrimer, acyclodextrin, a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof.

In addition, non-natural amino acid polypeptides may contain moietieswhich may be converted into other functional groups, such as, by way ofexample only, carbonyls, dicarbonyls or hydroxylamines. FIG. 63Aillustrates the chemical conversion of non-natural amino acidpolypeptides into carbonyl or dicarbonyl-containing non-natural aminoacid polypeptides, while FIG. 63B illustrates the chemical conversion ofnon-natural amino acid polypeptides into hydroxylamine-containingnon-natural amino acid polypeptides. The resulting carbonyl ordicarbonyl-containing non-natural amino acid polypeptides andhydroxylamine-containing non-natural amino acid polypeptides may be usedin or incorporated into any of the methods, compositions, techniques andstrategies for making, purifying, characterizing, and using non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides described herein. The chemicalconversion of chemical moieties into other functional groups, such as,by way of example only, carbonyls, di-carbonyls or hydroxylamines can beachieved using techniques and materials known to those of skill in theart, such as described, for example, in March, ADVANCED ORGANICCHEMISTRY 5^(th) Ed., (Wiley 2001); and Carey and Sundberg, ADVANCEDORGANIC CHEMISTRY 4^(th) Ed., Vols. A and B (Plenum 2000, 2001), (all ofwhich are incorporated by reference in their entirety).

Thus, by way of example only, a non-natural amino acid polypeptidecontaining any one of the following amino acids may be further modifiedusing the methods and compositions described herein:

(a)

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   J is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   each R″ is independently H, alkyl, substituted alkyl, or a    protecting group, or when more than one R″ group is present, two R″    optionally form a heterocycloalkyl;-   R₁ is H, an amino protecting group, resin; and-   R₂ is OH, an ester protecting group, resin;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   or the -A-B-J-R groups together form a bicyclic or tricyclic    cycloalkyl or heterocycloalkyl comprising at least one carbonyl    group, including a dicarbonyl group, protected carbonyl group,    including a protected dicarbonyl group, or masked carbonyl group,    including a masked dicarbonyl group;-   or the -J-R group together forms a monocyclic or bicyclic cycloalkyl    or heterocycloalkyl comprising at least one carbonyl group,    including a dicarbonyl group, protected carbonyl group, including a    protected dicarbonyl group, or masked carbonyl group, including a    masked dicarbonyl group;    (b)

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin; and-   R₂ is OH, an ester protecting group, resin;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   R₅ is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,    substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,    substituted aralkyl, -(alkylene or substituted alkylene)-ON(R″)₂,    -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or    substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″,    —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independently hydrogen,    alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,    substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,    substituted alkaryl, aralkyl, or substituted aralkyl;-   or R₅ is L-X, where X is a selected from the group consisting of a    label; a dye; a polymer; a water-soluble polymer; a derivative of    polyethylene glycol; a photocrosslinker; a cytotoxic compound; a    drug; an affinity label; a photoaffinity label; a reactive compound;    a resin; a second protein or polypeptide or polypeptide analog; an    antibody or antibody fragment; a metal chelator; a cofactor; a fatty    acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, -(alkylene or substituted    alkylene)-O—N═CR′-, -(alkylene or substituted    alkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene or    substituted alkylene)-S(O)_(k)-(alkylene or substituted    alkylene)-S—, -(alkylene or substituted alkylene)-S—S—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;    (c)

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   K is —NR₆R₇ or N═CR₆R₇;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin; and-   R₂ is OH, an ester protecting group, resin;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;    (d)

wherein;

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where each R′ is    independently H, alkyl, or substituted alkyl; or    (e)

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   M is —C(R₃)—,

where (a) indicates bonding to

-   -   the A group and (b) indicates bonding to respective carbonyl        groups, R₃ and R₄ are independently chosen from H, halogen,        alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl,        or R₃ and R₄ or two R₃ groups or two R₄ groups optionally form a        cycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl;

-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,    substituted alkyl, cycloalkyl, or substituted cycloalkyl;

-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and

-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.

In one aspect of the methods and compositions described herein arecompositions that include at least one polypeptide with at least one,including but not limited to, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten or more non-natural amino acids that havebeen post-translationally modified. The post-translationally-modifiednon-natural amino acids can be the same or different, including but notlimited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more different sites in the polypeptide thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more different post-translationally-modified non-naturalamino acids. In another aspect, a composition includes a polypeptidewith at least one, but fewer than all, of a particular amino acidpresent in the polypeptide is substituted with thepost-translationally-modified non-natural amino acid. For a givenpolypeptide with more than one post-translationally-modified non-naturalamino acids, the post-translationally-modified non-natural amino acidscan be identical or different (including but not limited to, thepolypeptide can include two or more different types ofpost-translationally-modified non-natural amino acids, or can includetwo of the same post-translationally-modified non-natural amino acid).For a given polypeptide with more than two post-translationally-modifiednon-natural amino acids, the post-translationally-modified non-naturalamino acids can be the same, different or a combination of a multiplepost-translationally-modified non-natural amino acid of the same kindwith at least one different post-translationally-modified non-naturalamino acid.

A. Methods for Post-Translationally Modifying Non-Natural Amino AcidPolypeptides: Reactions of Carbonyl-Containing Non-Natural Amino Acidswith Hydroxylamine-Containing Reagents

The sidechains of the naturally occurring amino acids lack highlyelectrophilic sites. Therefore, the incorporation of an unnatural aminoacid with an electrophile-containing sidechain, including, by way ofexample only, an amino acid containing a carbonyl or dicarbonyl groupsuch as ketones or aldehydes, makes possible the site-specificderivatization of this sidechain via nucleophilic attack of the carbonylor dicarbonyl group. In the instance where the attacking nucleophile isa hydroxylamine, an oxime-derivatized protein will be generated. Themethods for derivatizing and/or further modifying may be conducted witha polypeptide that has been purified prior to the derivatization step orafter the derivatization step. In addition, the methods for derivatizingand/or further modifying may be conducted with on synthetic polymers,polysaccharides, or polynucleotides which have been purified before orafter such modifications. Further, the derivatization step can occurunder mildly acidic to slightly basic conditions, including by way ofexample, between a pH of about 2-8, or between a pH of about 4-8.

A polypeptide-derivatizing method based upon the reaction of carbonyl-or dicarbonyl-containing polypeptides with a hydroxylamine-substitutedmolecule has distinct advantages. First, hydroxylamines undergocondensation with carbonyl- or dicarbonyl-containing compounds in a pHrange of 2-8 (and in further embodiments in a pH range of 4-8) togenerate oxime adducts. Under these conditions, the sidechains of thenaturally occurring amino acids are unreactive. Second, such selectivechemistry makes possible the site-specific derivatization of recombinantproteins: derivatized proteins can now be prepared as definedhomogeneous products. Third, the mild conditions needed to effect thereaction of the hydroxylamines described herein with the carbonyl- ordicarbonyl-containing polypeptides described herein generally do notirreversibly destroy the tertiary structure of the polypeptide(excepting, of course, where the purpose of the reaction is to destroysuch tertiary structure). Finally, although the hydroxylamine groupamino appears to be metabolized by E. coli, the condensation ofhydroxylamines with carbonyl- or dicarbonyl-containing moleculesgenerates oxime adducts which are stable under biological conditions.

By way of example only, the following non-natural amino acids are thetype of carbonyl- or dicarbonyl-containing amino acids that are reactivewith the hydroxylamine-containing reagents described herein that can beused to further modify carbonyl- or dicarbonyl-containing non-naturalamino acid polypeptides:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   J is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   each R″ is independently H, alkyl, substituted alkyl, or a    protecting group, or when more than one R″ group is present, two R″    optionally form a heterocycloalkyl;-   R₁ is H, an amino protecting group, resin; and-   R₂ is OH, an ester protecting group, resin;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   or the -A-B-J-R groups together form a bicyclic or tricyclic    cycloalkyl or heterocycloalkyl comprising at least one carbonyl    group, including a dicarbonyl group, protected carbonyl group,    including a protected dicarbonyl group, or masked carbonyl group,    including a masked dicarbonyl group;-   or the -J-R group together forms a monocyclic or bicyclic cycloalkyl    or heterocycloalkyl comprising at least one carbonyl group,    including a dicarbonyl group, protected carbonyl group, including a    protected dicarbonyl group, or masked carbonyl group, including a    masked dicarbonyl group.

In certain embodiments, compound of Formula (I) are reactive withhydroxylamines in aqueous solution under mildly acidic conditions. Incertain embodiments, such acidic conditions are pH 2 to 8.

By way of example only, for the aforementioned purposes, compounds ofFormula (I) include compounds having the structure:

wherein;

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and L is a bond, alkylene, substituted    alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where each    R′ is independently H, alkyl, or substituted alkyl.

By way of further example only, for the aforementioned purposes,compounds of Formula (I) include compounds having the structure ofFormula (XXXX):

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl substituted alkyl cycloalkyl, or substituted cycloalkyl,or R₃ and R₄ or two R₃ groups or two R₄ groups optionally form acycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl substituted alkyl cycloalkyl, or substituted    cycloalkyl;-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl    substituted alkyl cycloalkyl, or substituted cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.

The types of polypeptides that comprise such carbonyl- ordicarbonyl-containing non-natural amino acids is practically unlimitedas long as the carbonyl- or dicarbonyl-containing non-natural amino acidis located on the polypeptide so that the hydroxylamine reagent canreact with the carbonyl or dicarbonyl group and not create a resultingmodified non-natural amino acid that destroys the tertiary structure ofthe polypeptide (excepting, of course, if such destruction is thepurpose of the reaction).

By way of example only, the following hydroxylamine-containing reagentsare the type of hydroxylamine-containing reagents that are reactive withthe carbonyl- or dicarbonyl-containing non-natural amino acids describedherein and can be used to further modify carbonyl- ordicarbonyl-containing non-natural amino acid polypeptides:

[X-L]_(n)-L₁-W  (XIX)

wherein:

-   each X is independently H, alkyl substituted alkyl alkenyl,    substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,    substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,    polyalkylene oxide, substituted polyalkylene oxide, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,    substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or    substituted alkylene)-ON(R″)₂, -(alkylene or substituted    alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or    substituted aryl), —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each    R″ is independently hydrogen, alkyl substituted alkyl alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl;-   or each X is independently selected from the group consisting of a    label; a dye; a polymer; a water-soluble polymer; a derivative of    polyethylene glycol; a photocrosslinker; a cytotoxic compound; a    drug; an affinity label; a photoaffinity label; a reactive compound;    a resin; a second protein or polypeptide or polypeptide analog; an    antibody or antibody fragment; a metal chelator; a cofactor; a fatty    acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof;-   each L is independently selected from the group consisting of    alkylene, substituted alkylene, alkenylene, substituted alkenylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, -(alkylene or substituted alkylene)NR′C(O)O-(alkylene or    substituted alkylene)-, —O—CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —N(R′)C(O)O-(alkylene or substituted alkylene)-, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or substituted    alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)-;-   L₁ is optional, and when present, is —C(R′)_(p)—NR′ —C(O)O-(alkylene    or substituted alkylene)- where p is 0, 1, or 2;-   each R′ is independently H, alkyl, or substituted alkyl;-   W is N(R₈)₂, where each R₈ is independently H or an amino protecting    group; and n is 1 to 3;    provided that L-L₁-W together provide at least one hydroxylamine    group capable of reacting with a carbonyl (including a dicarbonyl)    group on a non-natural amino acid or a “modified or unmodified”    non-natural amino acid polypeptide.

In certain embodiments of compounds of Formula (XIX), X is a polymercomprising alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy,substituted alkylalkoxy, polyalkylene oxide, substituted polyalkyleneoxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl. Incertain embodiments of compounds of Formula (XIX), X is a polymercomprising polyalkylene oxide or substituted polyalkylene oxide. Incertain embodiments of compounds of Formula (XIX), X is a polymercomprising [(alkylene or substituted alkylene)-O-(hydrogen, alkyl, orsubstituted alkyl)]_(x), wherein x is from 20-10,000. In certainembodiments of compounds of Formula (XIX), X is m-PEG having a molecularweight ranging from 2 to 40 KDa. In certain embodiments of compounds ofFormula (XIX), X is a biologically active agent selected from the groupconsisting of a peptide, protein, enzyme, antibody, drug, dye, lipid,nucleoside, oligonucleotide, cell, virus, liposome, microparticle, andmicelle. In certain embodiments of compounds of Formula (XIX), X is adrug selected from the group consisting of an antibiotic, fungicide,anti-viral agent, anti-inflammatory agent, anti-tumor agent,cardiovascular agent, anti-anxiety agent, hormone, growth factor, andsteroidal agent. In certain embodiments of compounds of Formula (XIX), Xis a an enzyme selected from the group consisting of horseradishperoxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase.In certain embodiments of compounds of Formula (XIX), X is a detectablelabel selected from the group consisting of a fluorescent,phosphorescent, chemiluminescent, chelating, electron dense, magnetic,intercalating, radioactive, chromophoric, and energy transfer moiety. Incertain embodiments of compounds of Formula (XIX), L is selected fromthe group consisting of —N(R′)CO-(alkylene or substituted alkylene)-,—CON(R′)-(alkylene or substituted alkylene)-, —N(R′)C(O)N(R′)-(alkyleneor substituted alkylene)-, —O—CON(R′)-(alkylene or substitutedalkylene)-, —O-(alkylene or substituted alkylene)-, —C(O)N(R′)—, and—N(R′)C(O)O-(alkylene or substituted alkylene)-.

In certain embodiments of compounds of Formula (XIX), are compoundshaving the structure of Formula (XX):

X-L-O—NH₂  (XX).

In certain embodiments of compounds of Formula (XX), are compoundsselected from the group consisting of:

wherein other embodiments such m-PEG or PEG groups have a molecularweight ranging from 5 to 30 kDa.

In certain embodiments of compounds of Formula (XIX), are compoundshaving the structure of Formula (XXI):

In certain embodiments of compounds of Formula (XXI), are compoundsselected from the group consisting of:

In certain embodiments of compounds of Formula (XIX), are compoundshaving the structure of Formula (XXII):

In certain embodiments of compounds of Formula (XXII), L is -(alkyleneor substituted alkylene)-N(R′)C(O)O-(alkylene or substituted alkylene)-.In certain embodiments of compounds of Formula (XXII), are compoundshaving the structure of Formula (XXIII):

wherein other embodiments of compounds of Formula (XXII) such m-PEGgroups have a molecular weight ranging from 5 to 30 kDa.

In certain embodiments of compounds of Formula (XIX), are compoundshaving the structure of Formula (XXIV):

In certain embodiments of compounds of Formula (XXIV), L is -(alkyleneor substituted alkylene)-N(R′)C(O)O-(alkylene or substituted alkylene)-or —N(R′)C(O)O-(alkylene or substituted alkylene)-. In certainembodiments of compounds of Formula (XXIV), are compounds having thestructure of Formula (XXV):

wherein other embodiments of compounds of Formula (XXIV) such m-PEGgroups have a molecular weight ranging from 5 to 30 kDa.

In certain embodiments of compounds of Formula (XIX), are compoundshaving the structure of Formula (XXVI):

wherein each R₁₀ is independently H or an amino protecting group.In certain embodiments of compounds of Formula (XXVI), the polyalkyleneoxide is PEG. In other embodiments of compounds of Formula (XXVI), thePEG group has a molecular weight ranging from 5 to 30 kDa. In anotherembodiment of compounds of Formula (XXVI) is the compound correspondingto:

Three illustrative embodiments of methods for coupling a hydroxylamineto a carbonyl-containing non-natural amino acid on a polypeptide arepresented in FIG. 7. In these illustrative embodiments, ahydroxylamine-derivatized reagent is added to a buffered solution (pH2-8) of a carbonyl-containing non-natural amino acid polypeptide. Thereaction proceeds at the ambient temperature for hours to days. Toaccelerate the conjugation, additives such as those presented in FIG. 8are added; such compounds are also known herein as accelerants. Incertain embodiments, the accelerants or additives are capable of basecatalysis. The resulting oxime-containing non-natural amino acidpolypeptide is purified by HPLC, FPLC or size-exclusion chromatography.

In one embodiment, multiple linker chemistries can reactsite-specifically with a carbonyl- or dicarbonyl-substituted non-naturalamino acid polypeptide. In one embodiment, the linker methods describedherein utilize linkers containing the hydroxylamine functionality on atleast one linker termini (mono, bi- or multi-functional). Thecondensation of a hydroxylamine-derivatized linker with aketo-substituted protein generates a stable oxime linkage. Bi- and/ormulti-functional linkers (e.g., hydroxylamine with one, or more, otherlinking chemistries) allow the site-specific connection of differentmolecules (e.g., other proteins, polymers or small molecules) to thenon-natural amino acid polypeptide, while mono-functional linkers(hydroxylamine-substituted on all termini) facilitate the site-specificdimer- or oligomerization of the non-natural amino acid polypeptide. Bycombining this linker strategy with the in vivo translation technologydescribed herein, it becomes possible to specify the three-dimensionalstructures of chemically-elaborated proteins.

In certain embodiments are methods for derivatizing a polypeptidecomprising amino acids of Formulas I-XVIII, XXX-XXXIV(A&B), orXXXX-XXXXIII, including any sub-formulas or specific compounds that fallwithin the scope of Formulas I-XVIII, XXX-XXXIV(A&B), or XXXX-XXXXIII,wherein the method comprises contacting the polypeptide comprising atleast one amino acid of Formula I-XVIII, XXX-XXXIV(A&B), or XXXX-XXXXIIIwith a reagent of Formula (XIX). In certain embodiments the polypeptideis purified prior to or after contact with the reagent of Formula (XIX).In other embodiments are resulting derivatized polypeptide comprises atleast one oxime containing amino acid corresponding to Formula (XI),while in other embodiments such derivatized polypeptides are stable inaqueous solution for at least 1 month under mildly acidic conditions. Inother embodiments such derivatized polypeptides are stable for at least2 weeks under mildly acidic conditions. In other embodiments suchderivatized polypeptides are stable for at least 5 days under mildlyacidic conditions. In other embodiments such conditions are pH 2 to 8.In certain embodiments the tertiary structure of the derivatizedpolypeptide is preserved. In other embodiments such derivatization ofpolypeptides further comprises ligating the derivatized polypeptide toanother polypeptide. In other embodiments such polypeptides beingderivatized are homologous to a therapeutic protein selected from thegroup consisting of: alpha-1 antitrypsin, angiostatin, antihemolyticfactor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor,atrial natriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765,NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4,MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

In certain embodiments are methods for producing a polypeptide dimer,wherein the method comprises: (i) derivatizing a first polypeptidecomprising an amino acid of Formula (I) with a reagent of Formula(XXVI), and (ii) contacting the resulting derivatized protein of step(i) with a second protein comprising an amino acid of Formula (I),thereby forming a dimer comprising the first polypeptide and the secondpolypeptide. In other embodiments are methods for producing apolypeptide dimer, wherein the first polypeptide and the secondpolypeptide comprise an amino acid of corresponding to Formula (II). Incertain embodiments the polypeptides are purified prior to or aftercontact with the reagent of Formula (XXVI). In other embodiments theresulting derivatized protein of step (i) comprises at least one oximecontaining amino acid corresponding to Formula (XXVIII):

B. Methods for Post-Translationally Modifying Non-Natural Amino AcidPolypeptides: Reactions of Oxime-Containing Non-Natural Amino Acids withCarbonyl-Containing Reagents

A protein-derivatizing method based upon the exchange reaction of anoxime-containing protein with a carbonyl- or dicarbonyl-substitutedmolecule has distinct advantages. First, studies indicate that aminoacid-based oxime adducts undergo oxime exchange by equilibration with amore reactive carbonyl- or dicarbonyl-containing compound than the oneused to generate the original oxime. This exchange reaction occurs in apH range of 4-8: under these conditions, the side-chains of thenaturally occurring amino acids are unreactive. Thus, a general methodfor the preparation of carbonyl- or dicarbonyl-substituted moleculessuitable for reaction with oxime-containing proteins can provide accessto a wide variety of site-specifically derivatized proteins. In thecontext of this in vivo translation technology, a general method toprepare carbonyl- or dicarbonyl-substituted versions of those moleculesthat are typically used to derivatize proteins (including, by way ofexample only, hydrophilic polymers such as polyethylene glycol) arevaluable and will provide access to a wide variety of site-specificallyderivatized non-natural amino acid polypeptides. Second, such selectivechemistry makes possible the site-specific derivatization of recombinantproteins: derivatized proteins can now be prepared as definedhomogeneous products. Third, the mild conditions needed to affect theexchange reactions described herein generally do not irreversiblydestroy the tertiary structure of the polypeptide (excepting, of course,where the purpose of the reaction is to destroy such tertiarystructure). Finally, the exchange reactions generate new oxime adductswhich are stable under biological conditions.

By way of example only, the following non-natural amino acids are thetype of oxime-containing amino acids that are reactive with thecarbonyl- or dicarbonyl-containing reagents described herein that can beused to create new oxime-containing non-natural amino acid polypeptides:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   R₅ is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,    substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,    substituted aralkyl, -(alkylene or substituted alkylene)-ON(R″)₂,    -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or    substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″,    —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independently hydrogen,    alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,    substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,    substituted alkaryl, aralkyl, or substituted aralkyl;-   or R₅ is L-X, where X is a selected from the group consisting of a    label; a dye; a polymer; a water-soluble polymer; a derivative of    polyethylene glycol; a photocrosslinker; a cytotoxic compound; a    drug; an affinity label; a photoaffinity label; a reactive compound;    a resin; a second protein or polypeptide or polypeptide analog; an    antibody or antibody fragment; a metal chelator; a cofactor; a fatty    acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, -(alkylene or substituted    alkylene)-O—N═CR′-, -(alkylene or substituted    alkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene or    substituted alkylene)-S(O)_(k)-(alkylene or substituted    alkylene)-S—, -(alkylene or substituted alkylene)-S—S—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl.

By way of further example only, the following non-natural amino acidsare also the type of oxime-containing amino acids that are reactive withthe carbonyl- or dicarbonyl-containing reagents described herein thatcan be used to create new oxime-containing non-natural amino acidpolypeptides:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   each of R₆ and R₇ is independently selected from the group    consisting of H, alkyl, substituted alkyl, alkenyl, substituted    alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted    polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted    heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted    aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is    independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl; or R₆ or R₇ is L-X, where-   X is a selected from the group consisting of a label; a dye; a    polymer; a water-soluble polymer; a derivative of polyethylene    glycol; a photocrosslinker; a cytotoxic compound; a drug; an    affinity label; a photoaffinity label; a reactive compound; a resin;    a second protein or polypeptide or polypeptide analog; an antibody    or antibody fragment; a metal chelator; a cofactor; a fatty acid; a    carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof; and L is optional, and when present is a linker    selected from the group consisting of alkylene, substituted    alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,    —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,    —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl.

By way of example only, the following carbonyl- or dicarbonyl-containingreagents are the type of carbonyl- or dicarbonyl-containing reagentsthat are reactive with the oxime-containing non-natural amino acidsdescribed herein and can be used to effect exchange reactions to formnew oxime linkages and thus modify oxime-containing non-natural aminoacid polypeptides:

[X-L]_(n)-L₁-W  (XIX)

wherein:

-   each X is independently H, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,    substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,    polyalkylene oxide, substituted polyalkylene oxide, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,    substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or    substituted alkylene)-ON(R″)₂, -(alkylene or substituted    alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or    substituted aryl), —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each    R″ is independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl;-   or each X is independently selected from the group consisting of a    label; a dye; a polymer; a water-soluble polymer; a derivative of    polyethylene glycol; a photocrosslinker; a cytotoxic compound; a    drug; an affinity label; a photoaffinity label; a reactive compound;    a resin; a second protein or polypeptide or polypeptide analog; an    antibody or antibody fragment; a metal chelator; a cofactor; a fatty    acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof;-   each L is independently selected from the group consisting of    alkylene, substituted alkylene, alkenylene, substituted alkenylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, -(alkylene or substituted alkylene)NR′C(O)O-(alkylene or    substituted alkylene)-, —O—CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —N(R′)C(O)O-(alkylene or substituted alkylene)-, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or substituted    alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)-;-   L₁ is optional, and when present, is —C(R′)_(p)—NR′ —C(O)O-(alkylene    or substituted alkylene)- where p is 0, 1, or 2;-   each R′ is independently H, alkyl, or substituted alkyl;-   W is —C(═O)R₉, where R₉ is H or OR′; and n is 1 to 3;    or wherein L-L₁-W together forms a monocyclic or bicyclic cycloalkyl    or heterocycloalkyl comprising at least one carbonyl group,    including a dicarbonyl group, protected carbonyl group, including a    protected dicarbonyl group, or masked carbonyl group, including a    masked dicarbonyl group;    provided that L-L₁-W together provide at least one carbonyl group    (including a dicarbonyl group) capable of undergoing an oxime    exchange reaction with an oxime group on a non-natural amino acid or    a “modified or unmodified” non-natural amino acid polypeptide.

Two illustrative embodiments of methods for effect an oxime exchangereaction between an oxime-containing amino acid on a polypeptide and acarbonyl-containing reagent are presented in FIG. 9. In theseillustrative embodiments, a carbonyl-containing reagent is added to abuffered solution (pH 2-8) of an oxime-containing non-natural amino acidpolypeptide. The reaction proceeds at the ambient temperature for hoursto days. The modified oxime-containing non-natural amino acidpolypeptide is purified by HPLC, FPLC or size-exclusion chromatography.

In one embodiment, multiple linker chemistries can reactsite-specifically with an oxime-substituted non-natural amino acidpolypeptide. In one embodiment, the linker methods described hereinutilize linkers containing the carbonyl or dicarbonyl functionality onat least one linker termini (mono, bi- or multi-functional). Thecondensation of a carbonyl- or dicarbonyl-derivatized linker with anoxime-substituted non-natural amino acid polypeptide generates a newstable oxime linkage. Bi- and/or multi-functional linkers (e.g.,carbonyl or dicarbonyl with one, or more, other linking chemistries)allow the site-specific connection of different molecules (e.g., otherproteins, polymers or small molecules) to the non-natural amino acidpolypeptide, while mono-functional linkers (carbonyl- ordicarbonyl-substituted on all termini) facilitate the site-specificdimer- or oligomerization of the non-natural amino acid polypeptide. Bycombining this linker strategy with the in vivo translation technologydescribed herein, it becomes possible to specify the three-dimensionalstructures of chemically-elaborated proteins.

C Methods for Post-Translationally Modifying Non-Natural Amino AcidPolypeptides: Reactions of Hydroxylamine-Containing Non-Natural AminoAcids with Carbonyl-Containing Reagents

The post-translational modification techniques and compositionsdescribed above may also be used with hydroxylamine-containingnon-natural amino acids reacting with carbonyl- or dicarbonyl-containingreagents to produce modified oxime-containing non-natural amino acidpolypeptides.

A protein-derivatizing method based upon the reaction of ahydroxylamine-containing protein with a carbonyl- ordicarbonyl-substituted molecule has distinct advantages. First,hydroxylamines undergo condensation with carbonyl- ordicarbonyl-containing compounds in a pH range of 4-8 to generate oximeadducts. Under these conditions, the sidechains of the naturallyoccurring amino acids are unreactive. Second, such selective chemistrymakes possible the site-specific derivatization of recombinant proteins:derivatized proteins can now be prepared as defined homogeneousproducts. Third, the mild conditions needed to effect the reaction ofthe carbonyl- or dicarbonyl-containing reagents described herein withthe hydroxylamine-containing polypeptides described herein generally donot irreversibly destroy the tertiary structure of the polypeptide(excepting, of course, where the purpose of the reaction is to destroysuch tertiary structure). Finally, although the hydroxylamine groupamino appears to be metabolized by E. coli, the condensation ofcarbonyl- or dicarbonyl-containing reagents withhydroxylamine-containing amino acids generates oxime adducts which arestable under biological conditions.

By way of example only, the following non-natural amino acids are thetype of hydroxylamine-containing amino acids that are reactive with thecarbonyl- or dicarbonyl-containing reagents described herein that can beused to further modify hydroxylamine-containing non-natural amino acidpolypeptides:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   K is —NR₆R₇ or N═CR₆R₇;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl.

In certain embodiments of compounds of Formula (XIV) K is NH₂.

The types of polypeptides that comprise such hydroxylamine-containingnon-natural amino acids is practically unlimited as long as thehydroxylamine-containing non-natural amino acid is located on thepolypeptide so that the carbonyl- or dicarbonyl-containing reagent canreact with the hydroxylamine group and not create a resulting modifiednon-natural amino acid that destroys the tertiary structure of thepolypeptide (excepting, of course, if such destruction is the purpose ofthe reaction).

By way of example only, the following carbonyl- or dicarbonyl-containingreagents are the type of carbonyl- or dicarbonyl-containing reagentsthat are reactive with the hydroxylamine-containing non-natural aminoacids described herein and can be used to further modifyhydroxylamine-containing non-natural amino acid polypeptides:

[X-L]_(n)-L₁-W  (XIX)

wherein:

-   each X is independently H, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,    substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,    polyalkylene oxide, substituted polyalkylene oxide, aryl,    substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,    substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or    substituted alkylene)-ON(R″)₂, -(alkylene or substituted    alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or    substituted aryl), —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each    R″ is independently hydrogen, alkyl, substituted alkyl, alkenyl,    substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted    aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or    substituted aralkyl;-   or each X is independently selected from the group consisting of a    label; a dye; a polymer; a water-soluble polymer; a derivative of    polyethylene glycol; a photocrosslinker; a cytotoxic compound; a    drug; an affinity label; a photoaffinity label; a reactive compound;    a resin; a second protein or polypeptide or polypeptide analog; an    antibody or antibody fragment; a metal chelator; a cofactor; a fatty    acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense    polynucleotide; a saccharide, a water-soluble dendrimer, a    cyclodextrin, a biomaterial; a nanoparticle; a spin label; a    fluorophore, a metal-containing moiety; a radioactive moiety; a    novel functional group; a group that covalently or noncovalently    interacts with other molecules; a photocaged moiety; an actinic    radiation excitable moiety; a ligand; a photoisomerizable moiety;    biotin; a biotin analogue; a moiety incorporating a heavy atom; a    chemically cleavable group; a photocleavable group; an elongated    side chain; a carbon-linked sugar; a redox-active agent; an amino    thioacid; a toxic moiety; an isotopically labeled moiety; a    biophysical probe; a phosphorescent group; a chemiluminescent group;    an electron dense group; a magnetic group; an intercalating group; a    chromophore; an energy transfer agent; a biologically active agent;    a detectable label; a small molecule; an inhibitory ribonucleic    acid, a radionucleotide; a neutron-capture agent; a derivative of    biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an    abzyme, an activated complex activator, a virus, an adjuvant, an    aglycan, an allergan, an angiostatin, an antihormone, an    antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a    macromolecule, a mimotope, a receptor, a reverse micelle, and any    combination thereof;-   each L is independently selected from the group consisting of    alkylene, substituted alkylene, alkenylene, substituted alkenylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, -(alkylene or substituted alkylene)NR′C(O)O-(alkylene or    substituted alkylene)-, —O—CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —N(R′)C(O)O-(alkylene or substituted alkylene)-, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or substituted    alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)-;-   L₁ is optional, and when present, is —C(R′)_(p)—NR′ —C(O)O-(alkylene    or substituted alkylene)- where p is 0, 1, or 2;-   each R′ is independently H, alkyl, or substituted alkyl;-   W is -J-R, where J is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl; each R″ is independently H, alkyl, substituted alkyl, or    a protecting group, or when more than one R″ group is present, two    R″ optionally form a heterocycloalkyl; and n is 1 to 3;    provided that L-L₁-W together provide at least one carbonyl group    (including a dicarbonyl group) capable of reacting with an    hydroxylamine group on a non-natural amino acid or a “modified or    unmodified” non-natural amino acid polypeptide.

In certain embodiments of compounds of Formula (XIX), are compoundshaving the structure of Formula

An illustrative embodiment of methods for coupling a carbonyl-containingreagent to a hydroxylamine-containing non-natural amino acid on apolypeptide is presented in FIG. 10. In this illustrative embodiment, acarbonyl-derivatized reagent is added to a buffered solution (pH 2-8) ofa hydroxylamine-containing non-natural amino acid polypeptide. Thereaction proceeds at the ambient temperature for hours to days. Toaccelerate the conjugation, additives such as those presented in FIG. 8are added. The resulting oxime-containing non-natural amino acidpolypeptide is purified by HPLC, FPLC or size-exclusion chromatography.

In one embodiment, multiple linker chemistries can reactsite-specifically with a hydroxylamine-substituted non-natural aminoacid polypeptide. In one embodiment, the linker methods described hereinutilize linkers containing the carbonyl or dicarbonyl functionality onat least one linker termini (mono, bi- or multi-functional). Thecondensation of a carbonyl- or dicarbonyl-derivatized linker with ahydroxylamine-substituted protein generates a stable oxime linkage. Bi-and/or multi-functional linkers (e.g., carbonyl or dicarbonyl with one,or more, other linking chemistries) allow the site-specific connectionof different molecules (e.g., other proteins, polymers or smallmolecules) to the non-natural amino acid polypeptide, whilemono-functional linkers (carbonyl- or dicarbonyl-substituted on alltermini) facilitate the site-specific dimer- or oligomerization of thenon-natural amino acid polypeptide. By combining this linker strategywith the in vivo translation technology described herein, it becomespossible to specify the three-dimensional structures ofchemically-elaborated proteins.

In certain embodiments are methods for derivatizing a polypeptidecomprising amino acids of Formulas XIV-XVI, including any sub-formulasor specific compounds that fall within the scope of Formulas XIV-XVI,wherein the method comprises contacting the polypeptide comprising atleast one amino acid of Formula XIV-XVI with a reagent of Formula (XIX).In certain embodiments the polypeptide is purified prior to or aftercontact with the reagent of Formula (XIX). In other embodiments areresulting derivatized polypeptide comprises at least one oximecontaining amino acid corresponding to Formula (XXIX),

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower alkenylene, substituted lower alkenylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or    substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,    —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted    alkylene)-, —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or    substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted    alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,    —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,    —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   L is a linker independently selected from the group consisting of    alkylene, substituted alkylene, alkenylene, substituted alkenylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, -(alkylene or substituted alkylene)NR′C(O)O-(alkylene or    substituted alkylene)-, —O—CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —N(R′)C(O)O-(alkylene or substituted alkylene)-, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or substituted    alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide; each R₃ and R₄ is    independently H, halogen, lower alkyl, or substituted lower alkyl;    and-   X is independently a detectable label, biologically active agent, or    polymer.

In other embodiments such derivatized polypeptides are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In otherembodiments such derivatized polypeptides are stable for at least 2weeks under mildly acidic conditions. In other embodiments suchderivatized polypeptides are stable for at least 5 days under mildlyacidic conditions. In other embodiments such conditions are pH 2 to 8.In certain embodiments the tertiary structure of the derivatizedpolypeptide is preserved. In other embodiments such derivatization ofpolypeptides further comprises ligating the derivatized polypeptide toanother polypeptide. In other embodiments such polypeptides beingderivatized are homologous to a therapeutic protein selected from thegroup consisting of: alpha-1 antitrypsin, angiostatin, antihemolyticfactor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor,atrial natriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765,NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4,MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

D. Example of Adding Functionality: Macromolecular Polymers Coupled toNon-Natural Amino Acid Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug;an affinity label; a photoaffinity label; a reactive compound; a resin;a second protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin,a biomaterial; a nanoparticle; a spin label; a fluorophore, ametal-containing moiety; a radioactive moiety; a novel functional group;a group that covalently or noncovalently interacts with other molecules;a photocaged moiety; an actinic radiation excitable moiety; a ligand; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof. As an illustrative, non-limiting example of thecompositions, methods, techniques and strategies described herein, thefollowing description will focus on adding macromolecular polymers tothe non-natural amino acid polypeptide with the understanding that thecompositions, methods, techniques and strategies described thereto arealso applicable (with appropriate modifications, if necessary and forwhich one of skill in the art could make with the disclosures herein) toadding other functionalities, including but not limited to those listedabove.

A wide variety of macromolecular polymers and other molecules can becoupled to the non-natural amino acid polypeptides described herein tomodulate biological properties of the non-natural amino acid polypeptide(or the corresponding natural amino acid polypeptide), and/or providenew biological properties to the non-natural amino acid polypeptide (orthe corresponding natural amino acid polypeptide). These macromolecularpolymers can be coupled to the non-natural amino acid polypeptide viathe non-natural amino acid, or any functional substituent of thenon-natural amino acid, or any substituent or functional group added tothe non-natural amino acid.

Water soluble polymers can be coupled to the non-natural amino acidsincorporated into polypeptides (natural or synthetic), polynucleotides,poly saccharides or synthetic polymers described herein. The watersoluble polymers may be coupled via a non-natural amino acidincorporated in the polypeptide or any functional group or substituentof a non-natural amino acid, or any functional group or substituentadded to a non-natural amino acid. In some cases, the non-natural aminoacid polypeptides described herein comprise one or more non-naturalamino acid(s) coupled to water soluble polymers and one or morenaturally-occurring amino acids linked to water soluble polymers.Covalent attachment of hydrophilic polymers to a biologically activemolecule represents one approach to increasing water solubility (such asin a physiological environment), bioavailability, increasing serumhalf-life, increasing therapeutic half-life, modulating immunogenicity,modulating biological activity, or extending the circulation time of thebiologically active molecule, including proteins, peptides, andparticularly hydrophobic molecules. Additional important features ofsuch hydrophilic polymers include biocompatibility, lack of toxicity,and lack of immunogenicity. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable.

Examples of hydrophilic polymers include, but are not limited to:polyalkyl ethers and alkoxy-capped analogs thereof (e.g.,polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy orethoxy-capped analogs thereof, especially polyoxyethylene glycol, thelatter is also known as polyethylene glycol or PEG);polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyloxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkylacrylamides, and polyhydroxyalkyl acrylamides (e.g.,polyhydroxypropylmethacrylamide and derivatives thereof);polyhydroxyalkyl acrylates; polysialic acids and analogs thereof;hydrophilic peptide sequences; polysaccharides and their derivatives,including dextran and dextran derivatives, e.g., carboxymethyldextran,dextran sulfates, aminodextran; cellulose and its derivatives, e.g.,carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and itsderivatives, e.g., chitosan, succinyl chitosan, carboxymethylchitin,carboxymethylchitosan; hyaluronic acid and its derivatives; starches;alginates; chondroitin sulfate; albumin; pullulan and carboxymethylpullulan; polyaminoacids and derivatives thereof, e.g., polyglutamicacids, polylysines, polyaspartic acids, polyaspartamides; maleicanhydride copolymers such as: styrene maleic anhydride copolymer,divinylethyl ether maleic anhydride copolymer; polyvinyl alcohols;copolymers thereof; terpolymers thereof; mixtures thereof; andderivatives of the foregoing. The water soluble polymer may be anystructural form including but not limited to linear, forked or branched.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful. Multifunctional polymerderivatives include, but are not limited to, linear polymers having twotermini, each terminus being bonded to a functional group which may bethe same or different. In some embodiments, the water polymer comprisesa poly(ethylene glycol) moiety. The molecular weight of the polymer maybe of a wide range, including but not limited to, between about 100 Daand about 100,000 Da or more. The molecular weight of the polymer may bebetween about 100 Da and about 100,000 Da, including but not limited to,100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da,70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. The molecular weight of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and 50,000Da. In some embodiments, the molecular weight of the branched chain PEGis between about 1,000 Da and 40,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 5,000 Da and40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and 20,000 Da. Those of ordinaryskill in the art will recognize that the foregoing list forsubstantially water soluble backbones is by no means exhaustive and ismerely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inmethods and compositions described herein.

As described above, one example of a hydrophilic polymer ispoly(ethylene glycol), abbreviated PEG, which has been used extensivelyin pharmaceuticals, on artificial implants, and in other applicationswhere biocompatibility, lack of toxicity, and lack of immunogenicity areof importance. The polymer:polyeptide embodiments described herein willuse PEG as an example hydrophilic polymer with the understanding thatother hydrophilic polymers may be similarly utilized in suchembodiments.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). PEGis typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects.

The term “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to a non-natural amino acidpolypeptide by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group. The term PEG includes, but is not limited to,poly(ethylene glycol) in any of its forms, including bifunctional PEG,multiarmed PEG, derivatized PEG, forked PEG, branched PEG (with eachchain having a molecular weight of from about 1 kDa to about 100 kDa,from about 1 kDa to about 50 kDa, or from about 1 kDa to about 20 kDa),pendent PEG (i.e. PEG or related polymers having one or more functionalgroups pendent to the polymer backbone), or PEG with degradable linkagestherein. In one embodiment, PEG in which n is from about 20 to about2000 is suitable for use in the methods and compositions describedherein. In some embodiments, the water polymer comprises a polyethyleneglycol) moiety. The molecular weight of the PEG polymer may be of a widerange, including but not limited to, between about 100 Da and about100,000 Da or more. The molecular weight of the PEG polymer may bebetween about 100 Da and about 100,000 Da, including but not limited to,100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da,70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. The molecular weight of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and 50,000Da. In some embodiments, the molecular weight of the branched chain PEGis between about 1,000 Da and 40,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 5,000 Da and40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and 20,000 Da. A wide range of PEGmolecules are described in, including but not limited to, the ShearwaterPolymers, Inc. catalog, Nektar Therapeutics catalog, incorporated hereinby reference.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J.Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference intheir entirety.

In some cases, a PEG terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). Alternatively, the PEG canterminate with a reactive group, thereby forming a bifunctional polymer.Typical reactive groups can include those reactive groups that arecommonly used to react with the functional groups found in the 20 commonamino acids (including but not limited to, maleimide groups, activatedcarbonates (including but not limited to, p-nitrophenyl ester),activated esters (including but not limited to, N-hydroxysuccinimide,p-nitrophenyl ester) and aldehydes) as well as functional groups thatare inert to the 20 common amino acids but that react specifically withcomplementary functional groups present in non-natural amino acids(including but not limited to, oxime, carbonyl or dicarbonyl andhydroxylamine groups).

It is noted that the other end of the PEG, which is shown in the aboveformula by Y, will attach either directly or indirectly to a polypeptide(synthetic or natural), polynucleotide, polysaccharide or syntheticpolymer via a non-natural amino acid. When Y is a hydroxylamine group,then the hydroxylamine-containing PEG reagent can react with a carbonyl-or dicarbonyl-containing non-natural amino acid in a polypeptide to forma PEG group linked to the polypeptide via an oxime linkage. When Y is acarbonyl or dicarbonyl group, then the carbonyl- ordicarbonyl-containing PEG reagent can react with ahydroxylamine-containing non-natural amino acid in a polypeptide to forma PEG group linked to the polypeptide via an oxime linkage. When Y is acarbonyl or dicarbonyl group, then the carbonyl- ordicarbonyl-containing PEG reagent also can react with anoxime-containing non-natural amino acid in a polypeptide to form a PEGgroup linked to the polypeptide via a new oxime linkage. Examples ofappropriate reaction conditions, purification methods and reagents aredescribed throughout this specification and the accompanying Figures.For example, FIG. 7 presents three examples of the reaction of acarbonyl-containing non-natural amino acid polypeptide with ahydroxylamine-containing PEG reagent to form an oxime-containingnon-natural amino acid polypeptide linked to a PEG group. Further, FIG.9 presents two examples of the reaction of an oxime-containingnon-natural amino acid polypeptide with a carbonyl-containing PEGreagent to form a new oxime-containing non-natural amino acidpolypeptide linked to a PEG group. And FIG. 10 presents one example ofthe reaction of a hydroxylamine-containing non-natural amino acidpolypeptide with a carbonyl-containing PEG reagent to form anoxime-containing non-natural amino acid polypeptide linked to a PEGgroup.

By way of example only and not as a limitation on the types or classesof PEG reagents that may be used with the compositions, methods,techniques and strategies described herein, FIG. 11 presents furtherillustrative examples of hydroxylamine-containing PEG reagents that canreact with carbonyl-containing non-natural amino acid polypeptides toform oxime-containing non-natural amino acid polypeptides linked to thePEG group, as well as examples of carbonyl-containing PEG reagents thatcan react with react with oxime-containing non-natural amino acidpolypeptides or hydroxylamine-containing non-natural amino acidpolypeptides to form new oxime-containing non-natural amino acidpolypeptides linked to PEG groups. FIG. 12 presents four illustrativeexamples of synthetic methods for forming hydroxylamine-containing PEGreagents, or protected forms of hydroxylamine-containing PEG reagents,or masked forms of hydroxylamine-containing PEG reagents. FIG. 13presents an illustrative example of synthetic methods for formingamide-linked hydroxylamine-containing PEG reagents, or protected formsof amide-linked hydroxylamine-containing PEG reagents, or masked formsof amide-linked hydroxylamine-containing PEG reagents. FIG. 14 and FIG.15 present an illustrative examples of synthetic methods for formingcarbamate-linked hydroxylamine-containing PEG reagents, or protectedforms of carbamate-linked hydroxylamine-containing PEG reagents, ormasked forms of carbamate-linked hydroxylamine-containing PEG reagents.FIG. 16 presents illustrative examples of synthetic methods for formingsimple hydroxylamine-containing PEG reagents, or protected forms ofsimple hydroxylamine-containing PEG reagents, or masked forms of simplehydroxylamine-containing PEG reagents. Further, FIG. 17 presentsillustrative examples of hydroxylamine-containing reagents that havemultiple branches of linked PEG groups, and further shows the reactionof one such hydroxylamine-containing multi-PEG-branched reagents with acarbonyl-containing non-natural amino acid polypeptide to form anoxime-containing non-natural amino acid polypeptide with a linkedmulti-PEG-branched group.

Heterobifunctional derivatives are also particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In some embodiments, a strong nucleophile (including but not limited tohydroxylamine) can be reacted with an aldehyde or ketone group presentin a non-natural amino acid to form an oxime, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into thepolypeptide via a non-natural amino acid and used to reactpreferentially with a ketone or aldehyde group present in the watersoluble polymer. Generally, at least one terminus of the PEG molecule isavailable for reaction with the non-natural amino acid.

Thus, in some embodiments, the polypeptide comprising the non-naturalamino acid is linked to a water soluble polymer, such as polyethyleneglycol (PEG), via the side chain of the non-natural amino acid. Thenon-natural amino acid methods and compositions described herein providea highly efficient method for the selective modification of proteinswith PEG derivatives, which involves the selective incorporation ofnon-natural amino acids, including but not limited to, those amino acidscontaining functional groups or substituents not found in the 20naturally incorporated amino acids, into proteins in response to aselector codon and the subsequent modification of those amino acids witha suitably reactive PEG derivative. Known chemistry methodologies of awide variety are suitable for use with the non-natural amino acidmethods and compositions described herein to incorporate a water solublepolymer into the protein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is used in branched formsthat can be prepared by addition of ethylene oxide to various polyols,such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(m) in which R is derived from a core moiety,such as glycerol, glycerol oligomers, or pentaerythritol, and mrepresents the number of arms. Multi-armed PEG molecules, such as thosedescribed in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872;U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259, each ofwhich is incorporated by reference herein in its entirety, can also beused as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length. Yetanother branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown herein, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those skilled in the art that the term polyethyleneglycol or PEG represents or includes all the forms known in the artincluding but not limited to those disclosed herein. The molecularweight of the polymer may be of a wide range, including but not limitedto, between about 100 Da and about 100,000 Da or more. The molecularweight of the polymer may be between about 100 Da and about 100,000 Da,including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da,50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 50,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and 40,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 1,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 5,000Da and 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 10,000 Da and 40,000 Da.

In order to maximize the desired properties of PEG, the total molecularweight and hydration state of the PEG polymer or polymers attached tothe biologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

The methods and compositions described herein may be used to producesubstantially homogenous preparations of polymer:protein conjugates.“Substantially homogenous” as used herein means that polymer:proteinconjugate molecules are observed to be greater than half of the totalprotein. The polymer:protein conjugate has biological activity and thepresent “substantially homogenous” PEGylated polypeptide preparationsprovided herein are those which are homogenous enough to display theadvantages of a homogenous preparation, e.g., ease in clinicalapplication in predictability of lot to lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods described herein, and have amixture with a predetermined proportion of mono-polymer:proteinconjugates.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating hydrophilicpolymer:polypeptide/protein conjugates, the term “therapeuticallyeffective amount” further refers to an amount which gives an increase indesired benefit to a patient. The amount will vary from one individualto another and will depend upon a number of factors, including theoverall physical condition of the patient and the underlying cause ofthe disease, disorder or condition to be treated. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one skilled in the art using publicly available materials andprocedures.

The number of water soluble polymers linked to a “modified orunmodified” non-natural amino acid polypeptide (i.e., the extent ofPEGylation or glycosylation) described herein can be adjusted to providean altered (including but not limited to, increased or decreased)pharmacologic, pharmacokinetic or pharmacodynamic characteristic such asin vivo half-life. In some embodiments, the half-life of the polypeptideis increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent,two fold, five-fold, 10-fold, 50-fold, or at least about 100-fold overan unmodified polypeptide.

In one embodiment, a polypeptide comprising a carbonyl- ordicarbonyl-containing non-natural amino acid is modified with a PEGderivative that contains a terminal hydroxylamine moiety that is linkeddirectly to the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH,

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa). Themolecular weight of the polymer may be of a wide range, including butnot limited to, between about 100 Da and about 100,000 Da or more. Themolecular weight of the polymer may be between about 100 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da,700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of the polymer is between about 1,000 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 5,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and 40,000 Da.

In another embodiment, a polypeptide comprising a carbonyl- ordicarbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine moiety that is linked to the PEGbackbone by means of an amide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa). Themolecular weight of the polymer may be of a wide range, including butnot limited to, between about 100 Da and about 100,000 Da or more. Themolecular weight of the polymer may be between about 100 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da,700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of the polymer is between about 1,000 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 5,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and 40,000 Da.

In another embodiment, a polypeptide comprising a carbonyl- ordicarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydroxylamine moiety, with eachchain of the branched PEG having a MW ranging from 10-40 kDa and, inother embodiments, from 5-20 kDa. The molecular weight of the branchedpolymer may be of a wide range, including but not limited to, betweenabout 100 Da and about 100,000 Da or more. The molecular weight of thepolymer may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of the polymer is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand 40,000 Da. In some embodiments, the molecular weight of the polymeris between about 1,000 Da and 40,000 Da. In some embodiments, themolecular weight of the polymer is between about 5,000 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 10,000 Da and 40,000 Da.

In another embodiment, a polypeptide comprising a non-natural amino acidis modified with at least one PEG derivative having a branchedstructure. In some embodiments, the PEG derivatives containing ahydroxylamine group will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂C(O)—NH_CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000. Themolecular weight of the polymer may be between about 1,000 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In someembodiments, the molecular weight of the branched chain PEG is betweenabout 1,000 Da and 50,000 Da. In some embodiments, the molecular weightof the branched chain PEG is between about 1,000 Da and 40,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 5,000 Da and 40,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 5,000 Da and 20,000Da.

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), haemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-152 (1985)), all ofwhich are herein incorporated by reference in their entirety

If necessary, the PEGylated non-natural amino acid polypeptidesdescribed herein obtained from the hydrophobic chromatography can bepurified further by one or more procedures known to those skilled in theart including, but are not limited to, affinity chromatography; anion-or cation-exchange chromatography (using, including but not limited to,DEAE SEPHAROSE); chromatography on silica; reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography,metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), or extraction. Apparent molecular weight may beestimated by GPC by comparison to globular protein standards (Preneta AZ, PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris & Angal,Eds.) IRL Press 1989, 293-306). The purity of the non-natural amino acidpolypeptide:PEG conjugate can be assessed by proteolytic degradation(including but not limited to, trypsin cleavage) followed by massspectrometry analysis. Pepinsky R B., et. al., J. Pharmacol & Exp. Ther.297(3):1059-66 (2001).

A water soluble polymer linked to a non-natural amino acid of apolypeptide described herein can be further derivatized or substitutedwithout limitation.

E. Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the non-natural amino acidpolypeptides described herein to modulate the half-life in serum. Insome embodiments, molecules are linked or fused to the “modified orunmodified” non-natural amino acid polypeptides described herein toenhance affinity for endogenous serum albumin in an animal.

For example, in some cases, a recombinant fusion of a polypeptide and analbumin binding sequence is made. Exemplary albumin binding sequencesinclude, but are not limited to, the albumin binding domain fromstreptococcal protein G (see, e.g., Makrides et al., J. Pharmacol. Exp.Ther. 277(1):534-542 (1996) and Sjolander et al, J. Immunol Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277(38):35035-35043 (2002).

In other embodiments, the “modified or unmodified” non-natural aminoacid polypeptides described herein are acylated with fatty acids. Insome cases, the fatty acids promote binding to serum albumin. See, e.g.,Kurtzhals, et al, Biochem. J. 312:725-731 (1995).

In other embodiments, the “modified or unmodified” non-natural aminoacid polypeptides described herein are fused directly with serum albumin(including but not limited to, human serum albumin). Those of skill inthe art will recognize that a wide variety of other molecules can alsobe linked to non-natural amino acid polypeptides, modified orunmodified, as described herein, to modulate binding to serum albumin orother serum components.

F. Glycosylation of Non-Natural Amino Acid Polypeptides Described Herein

The methods and compositions described herein include polypeptidesincorporating one or more non-natural amino acids bearing saccharideresidues. The saccharide residues may be either natural (including butnot limited to, N-acetylglucosamine) or non-natural (including but notlimited to, 3-fluorogalactose). The saccharides may be linked to thenon-natural amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to the non-natural amino acid polypeptides either in vivo or invitro. In some embodiments, a polypeptide comprising a carbonyl- ordicarbonyl-containing non-natural amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-natural amino acid, the saccharide may be furtherelaborated by treatment with glycosyltransferases and other enzymes togenerate an oligosaccharide bound to the non-natural amino acidpolypeptide. See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703(2003).

G. Use of Linking Groups and Applications, Including Polypeptide Dimersand Multimers

In addition to adding functionality directly to the non-natural aminoacid polypeptide, the non-natural amino acid portion of the polypeptidemay first be modified with a multifunctional (e.g., bi-, tri, tetra-)linker molecule that then subsequently is further modified. That is, atleast one end of the multifunctional linker molecule reacts with atleast one non-natural amino acid in a polypeptide and at least one otherend of the multifunctional linker is available for furtherfunctionalization. If all ends of the multifunctional linker areidentical, then (depending upon the stoichiometric conditions)homomultimers of the non-natural amino acid polypeptide may be formed.If the ends of the multifunctional linker have distinct chemicalreactivities, then at least one end of the multifunctional linker groupwill be bound to the non-natural amino acid polypeptide and the otherend can subsequently react with a different functionality, including byway of example only: a label; a dye; a polymer; a water-soluble polymer;a derivative of polyethylene glycol; a photocrosslinker; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide, a water-soluble dendrimer, acyclodextrin, a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, a radionucleotide; a neutron-capture agent;a derivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter, an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof.

The multifunctional linker group has the general structure:

[X-L]_(n)-L₁-W  (XIX)

wherein:each X is independently NH₂, C(═O)R₉, —SR′ or -J-R′, where R₉ is H orOR′, where J is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl; each R″ is independently H, alkyl, substituted alkyl, or    a protecting group, or when more than one R″ group is present, two    R″ optionally form a heterocycloalkyl;-   each R′ is independently H, alkyl, or substituted alkyl;-   each L is independently selected from the group consisting of    alkylene, substituted alkylene, alkenylene, substituted alkenylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, -(alkylene or substituted alkylene)NR′C(O)O-(alkylene or    substituted alkylene)-, —O—CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —N(R′)C(O)O-(alkylene or substituted alkylene)-, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or substituted    alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)-;-   L₁ is optional, and when present, is —C(R′)_(p)—NR′ —C(O)O-(alkylene    or substituted alkylene)- where p is 0, 1, or 2;    -   W is NH₂, C(═O)R₉, —SR′ or J-R; and n is 1 to 3        provided that X and L-L₁-W together independently each provide        at least one of the following (a) a hydroxylamine group capable        of reacting with a carbonyl (including a dicarbonyl) group on a        non-natural amino acid or a “modified or unmodified” non-natural        amino acid polypeptide; (b) a carbonyl group (including a        dicarbonyl group) capable of reacting with an hydroxylamine        group on a non-natural amino acid or a “modified or unmodified”        non-natural amino acid polypeptide; or (c) a carbonyl group        (including a dicarbonyl group) capable of undergoing an exchange        reaction with an oxime group on a non-natural amino acid or a        “modified or unmodified” non-natural amino acid polypeptide.

FIG. 18 presents an illustrative, non-limiting example of the synthesisof a bifunctional homolinker in which the linker has two identical ends,i.e., hydroxylamine groups. Such a linker may be used to form ahomodimer of a carbonyl- or dicarbonyl-containing non-natural amino acidpolypeptide to form two oxime linkages. Alternatively, if one end ofsuch a linker is protected, then such a partially protected linker canbe used to bind the unprotected hydroxylamine end to a carbonyl- ordicarbonyl-containing non-natural amino acid polypeptide via an oximelinkage, leaving the other protected end available for further linkingreactions following deprotection. Alternatively, careful manipulation ofthe stoichiometry of the reagents may provide a similar result (aheterodimer), albeit a result in which the desired heterodimer willlikely be contaminated with some homodimer.

FIG. 19 presents illustrative, non-limiting examples of twomultifunctional heterolinkers in which each linker has more than onetype of terminal reactive group, i.e., hydroxylamine, oxime andthioester groups. Such a linker may be used to form a heterodimer of anon-natural amino acid polypeptide using the oxime-based chemistrydiscussed throughout this specification.

FIG. 20 presents a schematic illustrative, non-limiting example of theuse of a heterobifunctional linker to attach a PEG group to anon-natural amino acid polypeptide in a multi-step synthesis. In thefirst step, as depicted in this illustrative figure, acarbonyl-containing non-natural amino acid polypeptide reacts with ahydroxylamine-containing bifunctional linker to form a modifiedoxime-containing non-natural amino acid polypeptide. However, thebifunctional linker still retains a functional group (here illustratedby a shaped object) that is capable of reacting with a reagent withappropriate reactivity (illustrated in the figure by a matching shapedobject) to form a modified oxime-containing functionalized non-naturalamino acid polypeptide. In this particular illustrative figure, thefunctionalization is a PEG group, but may also include any of theaforementioned functionalities, or in this case of a tri- ortetra-functional linker, more than one type of functionality or multipletypes of the same functionality. FIG. 21 presents illustrative examplesof four types of linker groups used to link a hydroxylamine-containingnon-natural amino acid polypeptide to a PEG group. As before, the PEGfunctionality is provided for illustrative purposes only. Thus, thelinker groups described herein provide an additional means to furthermodify a non-natural amino acid polypeptide in a site-selective fashion.

The methods and compositions described herein also provide forpolypeptide combinations, such as homodimers, heterodimers,homomultimers, or heteromultimers (i.e., trimers, tetramers, etc.). Byway of example only, the following description focuses on the GHsupergene family members, however, the methods, techniques andcompositions described in this section can be applied to virtually anyother polypeptide which can provide benefit in the form of dimers andmultimers, including by way of example only: alpha-1 antitrypsin,angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein,atrial natriuretic factor, atrial natriuretic polypeptide, atrialpeptide, C—X—C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c,IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand,cytokine, CC chemokine, monocyte chemoattractant protein-1, monocytechemoattractant protein-2, monocyte chemoattractant protein-3, monocyteinflammatory protein-1 alpha, monocyte inflammatory protein-i beta,RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40ligand, c-kit ligand, collagen, colony stimulating factor (CSF),complement factor 5a, complement inhibitor, complement receptor 1,cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1,epidermal growth factor (EGF), epithelial neutrophil activating peptide,erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, FactorVIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin,four-helical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase,gonadotropin, growth factor, growth factor receptor, grf, hedgehogprotein, hemoglobin, hepatocyte growth factor (hGF), hirudin, humangrowth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor,LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

Thus, encompassed within the methods, techniques and compositionsdescribed herein are a GH supergene family member polypeptide containingone or more non-natural amino acids bound to another GH supergene familymember or variant thereof or any other polypeptide that is a non-GHsupergene family member or variant thereof, either directly to thepolypeptide backbone or via a linker. Due to its increased molecularweight compared to monomers, the GH supergene family member dimer ormultimer conjugates may exhibit new or desirable properties, includingbut not limited to different pharmacological, pharmacokinetic,pharmacodynamic, modulated therapeutic half-life, or modulated plasmahalf-life relative to the monomeric GH supergene family member. In someembodiments, the GH supergene family member dimers described herein willmodulate the dimerization of the GH supergene family member receptor. Inother embodiments, the GH supergene family member dimers or multimersdescribed herein will act as a GH supergene family member receptorantagonist, agonist, or modulator.

In some embodiments, the GH supergene family member polypeptides arelinked directly, including but not limited to, via an Asn-Lys amidelinkage or Cys-Cys disulfide linkage. In some embodiments, the linked GHsupergene family member polypeptides, and/or the linked non-GH supergenefamily member, will comprise different non-natural amino acids tofacilitate dimerization, including but not limited to, a first GHsupergene family member, and/or the linked non-GH supergene familymember, polypeptide comprising a ketone-containing non-natural aminoacid conjugated to a second GH supergene family member polypeptidecomprising a hydroxylamine-containing non-natural amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two GH supergene family member polypeptides, and/orthe linked non-GH supergene family member, are linked via a linker. Anyhetero- or homo-bifunctional linker can be used to link the two GHsupergene family member, and/or the linked non-GH supergene familymember, polypeptides, which can have the same or different primarysequence. In some cases, the linker used to tether the GH supergenefamily member, and/or the linked non-GH supergene family member,polypeptides together can be a bifunctional PEG reagent.

In some embodiments, the methods and compositions described hereinprovide for water-soluble bifunctional linkers that have a dumbbellstructure that includes: a) an azide, an alkyne, a hydrazine, ahydrazide, a hydroxylamine, or a carbonyl- or dicarbonyl-containingmoiety on at least a first end of a polymer backbone; and b) at least asecond functional group on a second end of the polymer backbone. Thesecond functional group can be the same or different as the firstfunctional group. The second functional group, in some embodiments, isnot reactive with the first functional group. The methods andcompositions described herein provide, in some embodiments,water-soluble compounds that comprise at least one arm of a branchedmolecular structure. For example, the branched molecular structure canbe dendritic.

In some embodiments, the methods and compositions described hereinprovide multimers comprising one or more GH supergene family memberformed by reactions with water soluble activated polymers that have thestructure:

R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X

wherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, aacetyl, or carbonyl- or dicarbonyl-containing moiety, and R is a cappinggroup, a functional group, or a leaving group that can be the same ordifferent as X. R can be, for example, a functional group selected fromthe group consisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

FIG. 22 presents an illustrative, non-limiting example of the use of alinker group described herein to form a homodimer of a non-natural aminoacid polypeptide described herein. In this illustrative example, acarbonyl-containing non-natural amino acid polypeptide is reacted with abifunctional linker group having two available hydroxylamine groupsunder conditions suitable for the formation of linked homodimeroxime-containing non-natural amino acid polypeptides. The methodpresented in the figure is not limited to carbonyl-containingnon-natural amino acid polypeptides coupled to hydroxylamine-containingbifunctional linkers. The non-natural amino acid polypeptide may furthercontain an oxime group that is capable of undergoing an exchangereaction with a carbonyl-containing multifunctional linker group to forma homomultimer linked by a structure containing multiple oxime groups,or the non-natural amino acid polypeptide may further contain ahydroxylamine group that is capable of undergoing a reaction with acarbonyl-containing multifunctional linker group to form a homomultimerlinked by a structure containing multiple oxime groups. Of course, thehomomultimer may be a homodimer, a homotrimer or a homotetramer.

FIG. 23 presents an illustrative, non-limiting example of the use of aheterofunctional linker group to form a heterodimer of polypeptides, inwhich at least one of the members of the heterodimer is a non-naturalamino acid polypeptide described herein and the other members areoptionally non-natural amino acid polypeptides as described herein,other types of non-natural amino acid polypeptides, ornaturally-occurring amino acid polypeptides. In the example presented inthis figure, the linker group contains two identical hydroxylaminegroups, by controlling the stoichiometry, temperature and otherparameters of the reaction, the dominant product from the reaction ofthe linker with a carbonyl-containing non-natural amino acid polypeptideis a modified oxime-containing non-natural amino acid polypeptideattached to a linker with an available hydroxylamine group. This lattergroup can further react with another carbonyl or dicarbonyl containingnon-natural amino acid polypeptide to form a bifunctional heterodimer ofoxime-containing non-natural amino acid polypeptides. Of course, thefunctional groups on the linker do not have to be identical, nor do theyhave to be hydroxylamine groups. Using the chemistry detailed throughoutthis specification, one of skill in the art could design a linker inwhich at least one functional group can form an oxime group with anon-natural amino acid polypeptide; the other functional groups on thelinker could utilize other known chemistry, including thenucleophile/electrophile based chemistry well known in the art oforganic chemistry.

H. Example of Adding Functionality: Easing the Isolation Properties of aPolypeptide

A naturally-occurring or non-natural amino acid polypeptide may bedifficult to isolate from a sample for a number of reasons, includingbut not limited to the solubility or binding characteristics of thepolypeptide. For example, in the preparation of a polypeptide fortherapeutic use, such a polypeptide may be isolated from a recombinantsystem that has been engineered to overproduce the polypeptide. However,because of the solubility or binding characteristics of the polypeptide,achieving a desired level of purity often proves difficult. The methods,compositions, techniques and strategies described herein provide asolution to this situation.

Using the methods, compositions, techniques and strategies describedherein, one of skill in the art can produce an oxime-containingnon-natural amino acid polypeptide that is homologous to the desiredpolypeptide, wherein the oxime-containing non-natural amino acidpolypeptide has improved isolation characteristics. In one embodiment, ahomologous non-natural amino acid polypeptide is producedbiosynthetically. In a further or additional embodiment, the non-naturalamino acid has incorporated into its structure one of the non-naturalamino acids described herein. In a further or additional embodiment, thenon-natural amino acid is incorporated at a terminal or internalposition and is further incorporated site specifically.

In one embodiment, the resulting non-natural amino acid, as producedbiosynthetically, already has the desired improved isolationcharacteristics. In further or additional embodiments, the non-naturalamino acid comprises an oxime linkage to a group that provides theimproved isolation characteristics. In further or additionalembodiments, the non-natural amino acid is further modified to form amodified oxime-containing non-natural amino acid polypeptide, whereinthe modification provides an oxime linkage to a group that provides theimproved isolation characteristics. In some embodiments, such a group isdirectly linked to the non-natural amino acid, and in other embodiments,such a group is linked via a linker group to the non-natural amino acid.In certain embodiments, such a group is connected to the non-naturalamino acid by a single chemical reaction, in other embodiments a seriesof chemical reactions is required to connect such a group to thenon-natural amino acid. Preferably, the group imparting improvedisolation characteristics is linked site specifically to the non-naturalamino acid in the non-natural amino acid polypeptide and is not linkedto a naturally occurring amino acid under the reaction conditionsutilized.

In further or additional embodiments the resulting non-natural aminoacid polypeptide is homologous to the GH supergene family members,however, the methods, techniques and compositions described in thissection can be applied to virtually any other polypeptide which canbenefit from improved isolation characteristics, including by way ofexample only: alpha-1 antitrypsin, angiostatin, antihemolytic factor,antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrialnatriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2,ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

In further or additional embodiments, the group imparting improvedisolation characteristics improves the water solubility of thepolypeptide; in other embodiments, the group improves the bindingproperties of the polypeptide; in other embodiments, the group providesnew binding properties to the polypeptide (including, by way of exampleonly, a biotin group or a biotin-binding group). In embodiments whereinthe group improves the water solubility of the polypeptide, the group isselected from the water soluble polymers described herein, including byway of example only, any of the PEG polymer groups described herein.

I. Example of Adding Functionality: Detecting the Presence of aPolypeptide

A naturally-occurring or non-natural amino acid polypeptide may bedifficult to detect in a sample (including an in vivo sample and an invitro sample) for a number of reasons, including but not limited to thelack of a reagent or label that can readily bind to the polypeptide. Themethods, compositions, techniques and strategies described hereinprovide a solution to this situation.

Using the methods, compositions, techniques and strategies describedherein, one of skill in the art can produce an oxime-containingnon-natural amino acid polypeptide that is homologous to the desiredpolypeptide, wherein the oxime-containing non-natural amino acidpolypeptide allows the detection of the polypeptide in an in vivo sampleand an in vitro sample. In one embodiment, a homologous non-naturalamino acid polypeptide is produced biosynthetically. In a further oradditional embodiment, the non-natural amino acid has incorporated intoits structure one of the non-natural amino acids described herein. In afurther or additional embodiment, the non-natural amino acid isincorporated at a terminal or internal position and is furtherincorporated site specifically.

In one embodiment, the resulting non-natural amino acid polypeptide, asproduced biosynthetically, already has the desired detectioncharacteristics. In further or additional embodiments, the non-naturalamino acid polypeptide comprises at least one non-natural amino acidselected from the group consisting of an oxime-containing non-naturalamino acid, a carbonyl-containing non-natural amino acid, and ahydroxylamine-containing non-natural amino acid. In other embodimentssuch non-natural amino acids have been biosynthetically incorporatedinto the polypeptide as described herein. In further or alternativeembodiments non-natural amino acid polypeptide comprises at least onenon-natural amino acid selected from amino acids of Formula I-XVIII,XXX-XXXIV(A&B), or XXXX-XXXXIII. In further or additional embodiments,the non-natural amino acid comprises an oxime linkage to a group thatprovides the improved detection characteristics. In further oradditional embodiments, the non-natural amino acid is further modifiedto form a modified oxime-containing non-natural amino acid polypeptide,wherein the modification provides an oxime linkage to a group thatprovides the improved detection characteristics. In some embodiments,such a group is directly linked to the non-natural amino acid, and inother embodiments, such a group is linked via a linker group to thenon-natural amino acid. In certain embodiments, such a group isconnected to the non-natural amino acid by a single chemical reaction,in other embodiments a series of chemical reactions is required toconnect such a group to the non-natural amino acid. Preferably, thegroup imparting improved detection characteristics is linked sitespecifically to the non-natural amino acid in the non-natural amino acidpolypeptide and is not linked to a naturally occurring amino acid underthe reaction conditions utilized.

In further or additional embodiments the resulting non-natural aminoacid polypeptide is homologous to the GH supergene family members,however, the methods, techniques and compositions described in thissection can be applied to virtually any other polypeptide which needs tobe detected in an in vivo sample and an in vitro sample, including byway of example only: alpha-1 antitrypsin, angiostatin, antihemolyticfactor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor,atrial natriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765,NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4,MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-1,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

In further or additional embodiments, the group imparting improveddetection characteristics is selected from the group consisting of alabel; a dye; an affinity label; a photoaffinity label; a spin label; afluorophore; a radioactive moiety; a moiety incorporating a heavy atom;an isotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; a chromophore; an energy transfer agent; a detectable label, andany combination thereof.

J. Example of Adding Functionality: Improving the Therapeutic Propertiesof a Polypeptide

A naturally-occurring or non-natural amino acid polypeptide will be ableto provide a certain therapeutic benefit to a patient with a particulardisorder, disease or condition. Such a therapeutic benefit will dependupon a number of factors, including by way of example only: the safetyprofile of the polypeptide, and the pharmacokinetics, pharmacologicsand/or pharmacodynamics of the polypeptide (e.g., water solubility,bioavailability, serum half-life, therapeutic half-life, immunogenicity,biological activity, or circulation time). In addition, it may beadvantageous to provide additional functionality to the polypeptide,such as an attached cytotoxic compound or drug, or it may be desirableto attach additional polypeptides to form the homo- and heteromultimersdescribed herein. Such modifications preferably do not destroy theactivity and/or tertiary structure of the original polypeptide. Themethods, compositions, techniques and strategies described hereinprovide solutions to these issues.

Using the methods, compositions, techniques and strategies describedherein, one of skill in the art can produce an oxime-containingnon-natural amino acid polypeptide that is homologous to the desiredpolypeptide, wherein the oxime-containing non-natural amino acidpolypeptide has improved therapeutic characteristics. In one embodiment,a homologous non-natural amino acid polypeptide is producedbiosynthetically. In a further or additional embodiment, the non-naturalamino acid has incorporated into its structure one of the non-naturalamino acids described herein. In a further or additional embodiment, thenon-natural amino acid is incorporated at a terminal or internalposition and is further incorporated site specifically.

In one embodiment, the resulting non-natural amino acid, as producedbiosynthetically, already has the desired improved therapeuticcharacteristics. In further or additional embodiments, the non-naturalamino acid comprises an oxime linkage to a group that provides theimproved therapeutic characteristics. In further or additionalembodiments, the non-natural amino acid is further modified to form amodified oxime-containing non-natural amino acid polypeptide, whereinthe modification provides an oxime linkage to a group that provides theimproved therapeutic characteristics. In some embodiments, such a groupis directly linked to the non-natural amino acid, and in otherembodiments, such a group is linked via a linker group to thenon-natural amino acid. In certain embodiments, such a group isconnected to the non-natural amino acid by a single chemical reaction,in other embodiments a series of chemical reactions is required toconnect such a group to the non-natural amino acid. Preferably, thegroup imparting improved therapeutic characteristics is linked sitespecifically to the non-natural amino acid in the non-natural amino acidpolypeptide and is not linked to a naturally occurring amino acid underthe reaction conditions utilized.

In further or additional embodiments the resulting non-natural aminoacid polypeptide is homologous to the GH supergene family members,however, the methods, techniques and compositions described in thissection can be applied to virtually any other polypeptide which canbenefit from improved therapeutic characteristics, including by way ofexample only: alpha-1 antitrypsin, angiostatin, antihemolytic factor,antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrialnatriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2,ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733,HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

In further or additional embodiments, the group imparting improvedtherapeutic characteristics improves the water solubility of thepolypeptide; in other embodiments, the group improves the bindingproperties of the polypeptide; in other embodiments, the group providesnew binding properties to the polypeptide (including, by way of exampleonly, a biotin group or a biotin-binding group). In embodiments whereinthe group improves the water solubility of the polypeptide, the group isselected from the water soluble polymers described herein, including byway of example only the PEG polymer groups. In further or additionalembodiments the group is a cytotoxic compound, whereas in otherembodiments the group is a drug. In further embodiments the linked drugor cytotoxic compound can be cleaved from the non-natural amino acidpolypeptide so as to deliver the drug or cytotoxic compound to a desiredtherapeutic location. In other embodiments, the group is a secondpolypeptide, including by way of example, an oxime-containingnon-natural amino acid polypeptide, further including by way of example,a polypeptide that has the same amino acid structure as the firstnon-natural amino acid polypeptide.

In further or additional embodiments, the oxime-containing non-naturalamino acid polypeptide is a modified oxime-containing non-natural aminoacid polypeptide. In further or additional embodiments, theoxime-containing non-natural amino acid polypeptide increases thebioavailability of the polypeptide relative to the homologousnaturally-occurring amino acid polypeptide. In further or additionalembodiments, the oxime-containing non-natural amino acid polypeptideincreases the safety profile of the polypeptide relative to thehomologous naturally-occurring amino acid polypeptide. In further oradditional embodiments, the oxime-containing non-natural amino acidpolypeptide increases the water solubility of the polypeptide relativeto the homologous naturally-occurring amino acid polypeptide. In furtheror additional embodiments, the oxime-containing non-natural amino acidpolypeptide increases the therapeutic half-life of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.In further or additional embodiments, the oxime-containing non-naturalamino acid polypeptide increases the serum half-life of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.In further or additional embodiments, the oxime-containing non-naturalamino acid polypeptide extends the circulation time of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.In further or additional embodiments, the oxime-containing non-naturalamino acid polypeptide modulates the activity of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.In further or additional embodiments, the oxime-containing non-naturalamino acid polypeptide modulates the immunogenicity of the polypeptiderelative to the homologous naturally-occurring amino acid polypeptide.

XI. Therapeutic Uses of Modified Polypeptides

For convenience, the “modified or unmodified” non-natural polypeptidesdescribed in this section have been described generically and/or withspecific examples. However, the “modified or unmodified” non-naturalpolypeptides described in this section should not be limited to just thegeneric descriptions or specific example provided in this section, butrather the “modified or unmodified” non-natural polypeptides describedin this section apply equally well to all “modified or unmodified”non-natural polypeptides comprising at least one amino acid which fallswithin the scope of Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII,including any sub-formulas or specific compounds that fall within thescope of Formulas I-XVIII, XXX-XXXIV(A&B), and XXXX-XXXXIII that aredescribed in the specification, claims and figures herein.

The “modified or unmodified” non-natural amino acid polypeptidesdescribed herein, including homo- and hetero-multimers thereof findmultiple uses, including but not limited to: therapeutic, diagnostic,assay-based, industrial, cosmetic, plant biology, environmental,energy-production, and/or military uses. As a non-limiting illustration,the following therapeutic uses of “modified or unmodified” non-naturalamino acid polypeptides are provided.

The “modified or unmodified” non-natural amino acid polypeptidesdescribed herein are useful for treating a wide range of disorders,conditions or diseases. Administration of the “modified or unmodified”non-natural amino acid polypeptide products described herein results inany of the activities demonstrated by commercially available polypeptidepreparations in humans. Average quantities of the “modified orunmodified” non-natural amino acid polypeptide product may vary and inparticular should be based upon the recommendations and prescription ofa qualified physician. The exact amount of the “modified or unmodified”non-natural amino acid polypeptide is a matter of preference subject tosuch factors as the exact type of condition being treated, the conditionof the patient being treated, as well as the other ingredients in thecomposition. The amount to be given may be readily determined by oneskilled in the art based upon therapy with the “modified or unmodified”non-natural amino acid polypeptide.

A. Administration and Pharmaceutical Compositions

The “modified or unmodified” non-natural amino acid polypeptidesdescribed herein, including homo- and hetero-multimers thereof findmultiple uses, including but not limited to: therapeutic, diagnostic,assay-based, industrial, cosmetic, plant biology, environmental,energy-production, and/or military uses. As a non-limiting illustration,the following therapeutic uses of “modified or unmodified” non-naturalamino acid polypeptides are provided.

The “modified or unmodified” non-natural amino acid polypeptidesdescribed herein are useful for treating a wide range of disorders.Administration of the “modified or unmodified” non-natural amino acidpolypeptide products described herein results in any of the activitiesdemonstrated by commercially available polypeptide preparations inhumans. Average quantities of the “modified or unmodified” non-naturalamino acid polypeptide product may vary and in particular should bebased upon the recommendations and prescription of a qualifiedphysician. The exact amount of the “modified or unmodified” non-naturalamino acid polypeptide is a matter of preference subject to such factorsas the exact type of condition being treated, the condition of thepatient being treated, as well as the other ingredients in thecomposition. The amount to be given may be readily determined by oneskilled in the art based upon therapy with the “modified or unmodified”non-natural amino acid polypeptide.

The non-natural amino acid polypeptides, modified or unmodified, asdescribed herein (including but not limited to, synthetases, proteinscomprising one or more non-natural amino acid, etc.) are optionallyemployed for therapeutic uses, including but not limited to, incombination with a suitable pharmaceutical carrier. Such compositions,for example, comprise a therapeutically effective amount of thenon-natural amino acid polypeptides, modified or unmodified, asdescribed herein, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are wellknown in the art and can be applied to administration of the non-naturalamino acid polypeptides, modified or unmodified, as described herein.

Therapeutic compositions comprising one or more of the non-natural aminoacid polypeptides, modified or unmodified, as described herein areoptionally tested in one or more appropriate in vitro and/or in vivoanimal models of disease, to confirm efficacy, tissue metabolism, and toestimate dosages, according to methods well known in the art. Inparticular, dosages can be initially determined by activity, stabilityor other suitable measures of non-natural to natural amino acidhomologues (including but not limited to, comparison of a polypeptidemodified to include one or more non-natural amino acids to a naturalamino acid polypeptide), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. Thenon-natural amino acid polypeptides, modified or unmodified, asdescribed herein, are administered in any suitable manner, optionallywith one or more pharmaceutically acceptable carriers. Suitable methodsof administering the non-natural amino acid polypeptides, modified orunmodified, as described herein, to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsdescribed herein.

The non-natural amino acid polypeptides described herein andcompositions comprising such polypeptides may be administered by anyconventional route suitable for proteins or peptides, including, but notlimited to parenterally, e.g. injections including, but not limited to,subcutaneously or intravenously or any other form of injections orinfusions. Polypeptide pharmaceutical compositions (including thevarious non-natural amino acid polypeptides described herein) can beadministered by a number of routes including, but not limited to oral,intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous,topical, sublingual, or rectal means. Compositions comprisingnon-natural amino acid polypeptides, modified or unmodified, asdescribed herein, can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art. The non-natural amino acid polypeptidesdescribed herein may be used alone or in combination with other suitablecomponents, including but not limited to, a pharmaceutical carrier.

The non-natural amino acid polypeptides, modified or unmodified, asdescribed herein, alone or in combination with other suitablecomponents, can also be made into aerosol formulations (i.e., they canbe “nebulized”) to be administered via inhalation. Aerosol formulationscan be placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, IFN, GH, G-CSF,GM-CSF, IFNs, interleukins, antibodies, and/or any otherpharmaceutically delivered protein), along with formulations in currentuse, provide preferred routes of administration and formulation for thenon-natural amino acid polypeptides, modified or unmodified, asdescribed herein.

The dose administered to a patient, in the context compositions andmethods described herein, is sufficient to have a beneficial therapeuticresponse in the patient over time. The dose is determined by theefficacy of the particular formulation, and the activity, stability orserum half-life of the non-natural amino acid polypeptides, modified orunmodified, employed and the condition of the patient, as well as thebody weight or surface area of the patient to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularformulation, or the like in a particular patient.

In determining the effective amount of the formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-non-natural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The pharmaceutical formulationsdescribed herein can supplement treatment conditions by any knownconventional therapy, including antibody administration, vaccineadministration, administration of cytotoxic agents, natural amino acidpolypeptides, nucleic acids, nucleotide analogues, biologic responsemodifiers, and the like.

For administration, the pharmaceutical formulations described herein areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the non-naturalamino acid polypeptides, modified or unmodified, at variousconcentrations, including but not limited to, as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Non-natural amino acid polypeptides, modified or unmodified, asdescribed herein, can be administered directly to a mammalian subject.Administration is by any of the routes normally used for introducing apolypeptide to a subject. The non-natural amino acid polypeptides,modified or unmodified, as described herein, include those suitable fororal, rectal, topical, inhalation (including but not limited to, via anaerosol), buccal (including but not limited to, sub-lingual), vaginal,parenteral (including but not limited to, subcutaneous, intramuscular,intradermal, intraarticular, intrapleural, intraperitoneal,inracerebral, intraarterial, or intravenous), topical (i.e., both skinand mucosal surfaces, including airway surfaces) and transdermaladministration, although the most suitable route in any given case willdepend on the nature and severity of the condition being treated.Administration can be either local or systemic. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampoulesand vials. The non-natural amino acid polypeptides, modified orunmodified, as described herein, can be prepared in a mixture in a unitdosage injectable form (including but not limited to, solution,suspension, or emulsion) with a pharmaceutically acceptable carrier. Thenon-natural amino acid polypeptides, modified or unmodified, asdescribed herein, can also be administered by continuous infusion(using, including but not limited to, minipumps such as osmotic pumps),single bolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which are hereinincorporated by reference in their entirety, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions described herein may comprise apharmaceutically acceptable carrier, excipient or stabilizer.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions(including optional pharmaceutically acceptable carriers, excipients, orstabilizers) for the non-natural amino acid polypeptides, modified orunmodified, described herein, (see, for example, in Remington: TheScience and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins, 1999)). Suitable carriersinclude buffers containing succinate, phosphate, borate, HEPES, citrate,imidazole, acetate, bicarbonate, and other organic acids; antioxidantsincluding but not limited to, ascorbic acid; low molecular weightpolypeptides including but not limited to those less than about 10residues; proteins, including but not limited to, serum albumin,gelatin, or immunoglobulins; hydrophilic polymers including but notlimited to, polyvinylpyrrolidone; amino acids including but not limitedto, glycine, glutamine, asparagine, arginine, histidine or histidinederivatives, methionine, glutamate, or lysine; monosaccharides,disaccharides, and other carbohydrates, including but not limited to,trehalose, sucrose, glucose, mannose, or dextrins; chelating agentsincluding but not limited to, EDTA; divalent metal ions including butnot limited to, zinc, cobalt, or copper; sugar alcohols including butnot limited to, mannitol or sorbitol; salt-forming counter ionsincluding but not limited to, sodium; and/or nonionic surfactants,including but not limited to Tween™ (including but not limited to, Tween80 (polysorbate 80) and Tween 20 (polysorbate 20), Pluronics™ and otherpluronic acids, including but not limited to, and other pluronic acids,including but not limited to, pluronic acid F68 (poloxamer 188), or PEG.Suitable surfactants include for example but are not limited topolyethers based upon poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO), or polypropyleneoxide)-poly(ethylene oxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO),or a combination thereof. PEO-PPO-PEO and PPO-PEO-PPO are commerciallyavailable under the trade names Pluronics™, R-Pluronics™, Tetronics™ andR-Tetronics™ (BASF Wyandotte Corp., Wyandotte, Mich.) and are furtherdescribed in U.S. Pat. No. 4,820,352 incorporated herein in its entiretyby reference. Other ethylene/polypropylene block polymers may besuitable surfactants. A surfactant or a combination of surfactants maybe used to stabilize PEGylated non-natural amino acid polypeptidesagainst one or more stresses including but not limited to stress thatresults from agitation. Some of the above may be referred to as “bulkingagents.” Some may also be referred to as “tonicity modifiers.”

The non-natural amino acid polypeptides, modified or unmodified, asdescribed herein, including those linked to water soluble polymers suchas PEG can also be administered by or as part of sustained-releasesystems. Sustained-release compositions include, including but notlimited to, semi-permeable polymer matrices in the form of shapedarticles, including but not limited to, films, or microcapsules.Sustained-release matrices include from biocompatible materials such aspoly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res., 15: 167-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982),ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 22, 547-556(1983), poly(ortho)esters, polypeptides, hyaluronic acid, collagen,chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,polysaccharides, nucleic acids, polyamino acids, amino acids such asphenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone and silicone. Sustained-releasecompositions also include a liposomally entrapped compound. Liposomescontaining the compound are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324.

Liposomally entrapped polypeptides can be prepared by methods describedin, e.g., DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A.,82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Composition and size of liposomes are wellknown or able to be readily determined empirically by one skilled in theart. Some examples of liposomes as described in, e.g., Park J W, et al.,Proc. Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D andPapahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998);Drummond D C, et al., Liposomal drug delivery systems for cancertherapy, in Teicher B (ed): CANCER DRUG DISCOVERY AND DEVELOPMENT(2002); Park J W, et al., Clin. Cancer Res. 8:1172-1181 (2002); NielsenU B, et al., Biochim. Biophys. Acta 1591(1-3):109-118 (2002); Mamot C,et al., Cancer Res. 63: 3154-3161 (2003).

The dose administered to a patient in the context of the compositions,formulations and methods described herein, should be sufficient to causea beneficial response in the subject over time. Generally, the totalpharmaceutically effective amount of the non-natural amino acidpolypeptides, modified or unmodified, as described herein, administeredparenterally per dose is in the range of about 0.01 μg/kg/day to about100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, of patient body weight,although this is subject to therapeutic discretion. The frequency ofdosing is also subject to therapeutic discretion, and may be morefrequent or less frequent than the commercially available productsapproved for use in humans. Generally, a polymer:polypeptide conjugate,including by way of example only, a PEGylated polypeptide, as describedherein, can be administered by any of the routes of administrationdescribed above.

EXAMPLES Example 1

This example details the synthesis of the carbonyl-containing amino acidpresented in FIG. 4. The carbonyl-containing non-natural amino acid wasproduced as described in FIG. 4.

Example 2

This example details the synthesis of the protectedhydroxylamine-containing amino acid presented in FIG. 5 a. The protectedhydroxylamine-containing non-natural amino acid was produced asdescribed in FIG. 5 a.

Example 3

This example details the synthesis of the hydroxylamine-containing aminoacid presented in FIG. 5 b. The hydroxylamine-containing non-naturalamino acid was produced as described in FIG. 5 b

Example 4

This example details the synthesis of the hydroxylamine-containing aminoacid presented in FIG. 5 c. The hydroxylamine-containing non-naturalamino acid was produced as described in FIG. 5 c.

Example 5

This example details the synthesis of the oxime-containing amino acidpresented in FIG. 5 d. The oxime-containing non-natural amino acid wasproduced as described in FIG. 5 d.

Example 6

This example details the synthesis of the oxime-containing amino acidpresented in FIG. 6 a. The oxime-containing non-natural amino acid wasproduced as described in FIG. 6 a.

Example 7

This example details the synthesis of the oxime-containing amino acidpresented in FIG. 6 b. The oxime-containing non-natural amino acid wasproduced as described in FIG. 6 b.

Example 8

This example details the synthesis of the oxime-containing amino acidpresented in FIG. 6 c. The oxime-containing non-natural amino acid wasproduced as described in FIG. 6 c.

Example 9

This example details the synthesis of the carbonyl-containing amino acidpresented in FIG. 24.

To a solution NaOH (40 mL, 25% vol.) at 0° C. was added ether (60 mL). Ablast shield was placed in front of the reaction flask. To the resultantmixture was added N-nitroso-N-methyl urea (6.0 g, 57.9 mmol) in 3portions over 3 minutes. The reaction was stirred at 0° C. for 10minutes. The diethyl ether and sodium hydroxide layers were then allowedto separate. The organic layer was added to the solution ofN-Boc-4-hydroxymethylphenylalanine (7.5 g, 25.4 mmol) in anhydrous THF(20 mL) potionwise (approximately 6 additions) over 5 minutes until thestarting material had completely disappeared (monitored by TLC). 5 propsof glacial acetic acid were then added to quench the reaction. After theorganic solvents were removed by rotary evaporation, ethyl acetate wasadded. The organic layer was washed successively with saturated NaHCO₃solution, H₂O and brine, then dried over anhydrous MgSO₄, filtered andconcentrated to yield the product (5.9 g, 75%) as a white powder.

To a stirred solution of alcohol (6.0 g, 19.4 mmol) and pyridine (12 mL,150 mmol) in CH₂Cl₂ (400 mL) at 0° C. was added Dess-Martin periodinane(14.2 g, 33.4 mmol). The mixture was stirred at room temperatureovernight. The reaction was then quenched with saturated aqueousNa₂S₂O₃—NaHCO₃ (1:1, 300 mL) and extracted with CH₂Cl₂. The organiclayers were combined and washed with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 1:100-1:1 hexane:EtOAc)afforded the aldehyde product (5.48 g, 92%) as a white solid.

To a solution of aldehyde (3.07 g, 10 mmol) in EtOH (40 mL) was addedacetic hydrazide (1.7 g, 20 mmol). The reaction mixture was stirred atroom temperature for 30 minutes and concentrated. To the residue wasadded H₂O (200 mL) followed by CH₂Cl₂. The organic layer was separatedand concentrated in vacuo. Purification of the residue by flashchromatography (silica, 3:7-1:9 hexane:EtOAc) yielded the product (3.29g, 90%) as a white solid.

To a solution of the above methyl ester (3.29 g, 9.1 mmol) in dioxane(10 mL) at 0° C. was added LiOH (10 mL, 1 N). The mixture was stirred atthe same temperature for 1 h and then quenched by the addition of citricacid (5 g) and diluted with H₂O. The mixture was extracted with EtOAc.The organic layer was washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered, and concentrated to afford a whitesolid (3.05 g, 96%).

To a solution of the above acid (3.02 g, 8.6 mmol) in CH₂Cl₂ (20 mL) at0° C. was added trifluoroacetic acid (20 mL). The reaction mixture wasstirred at 0° C. for 2 h and concentrated. To the residue was added MeOH(1 mL) followed by the addition of HCl (2.0 mL, 4 N in dioxane). Ether(200 mL) was then added to precipitate the product (2.07 g, 83%) as ayellow solid.

Example 10

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 25.

To a stirred solution of amine (10 g, 34 mmol) in DMF (70 mL) at 0° C.were added pyruvate acid (5 mL, 72 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 20 g,104 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 85 g, 71 mmol) andN,N-diisopropylethylamine (DIEA, 35 mL, 200 mmol). The mixture wasstirred at room temperature for 6 h and then quenched with aqueouscitric acid solution (5%, 500 mL) and extracted with EtOAc (500 mL). Theorganic layer was washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residuewas purified by flash chromatography (silica, 3:1-1:1 hexane:EtOAc) toafford product as a solid (4.78 g, 40%).

To a solution of the above methyl ester (2.96 g, 8.1 mmol) in dioxane(10 mL) at 0° C. was added LiOH (10 mL, 1 N). The mixture was stirred atthe same temperature for 3 hours. The reaction was then quenched withaqueous citric acid solution (5%) and diluted with EtOAc. The organiclayer was separated and washed successively with H₂O and brine, thendried over anhydrous Na₂SO₄, filtered, and concentrated to affordproduct as a yellow solid (2.87 g, 100%).

To a solution of the above acid (2.05 g, 5.9 mmol) in CH₂Cl₂ (10 mL) at0° C. was added trifluoroacetic acid (10 mL). The mixture was stirredfor 2 h and concentrated in vacuo. To the residue was added HCl (1 mL, 4N in dioxane) followed by ether (400 mL). The precipitate was collectedas a white solid (1.38 g, 82%).

Example 11

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 26.

To a solution of 4′-methylpropiophenone (20 g, 122 mmol) andN-bromosuccinimde (NBS, 23 g, 130 mmol) in benzene (300 mL) at 90° C.was added 2,2′-azobisisobutyronitrile (AIBN, 0.6 g, 3.6 mmol). Theresultant solution was heated to reflux overnight. The reaction was thencooled to room temperature. The brown solution was washed successivelywith H₂O and brine, then dried over anhydrous Na₂SO₄, filtered, andconcentrated in vacuo. The residue was crystallized from hexanes toafford product as a light yellow solid (27 g, 87%).

To a solution of EtONa (14.5 g, 203 mmol) in EtOH (400 mL) at 0° C. wasadded diethyl acetamidomalonate (39 g, 180 mmol) followed by thesolution of the above bromide (27 g, 119 mmol) in EtOH (100 mL). Theresultant mixture was heated to reflux for 1 h and quenched with citricacid (30 g) and diluted with H₂O (300 mL). After most solvent wasremoved in vacuo, the residue was extracted with EtOAc. The organiclayer was washed successively with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by flash chromatography (silica, 10:1-3:1 hexane:EtOAc) toafford product (37 g, 88%) as a yellow solid.

To a solution of the ketone (5 g, 13.8 mmol) in ether (100 mL) at 0° C.was added Br₂ (0.8 mL, 15.6 mmol). The mixture was stirred at roomtemperature for 3 h and then quenched with saturated aqueous NaHCO₃. Themixture was extracted with Et₂O. The organic layer was washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered, and concentrated in vacuo to afford product as a yellow solid(5.4 g, 88%) which was directly used for the next step with furtherpurification.

To the solution of α-bromo ketone (5.4 g, 12.2 mmol) and Na₂CO₃ (2.0 g,18.9 mmol) in DMSO (20 mL) was added KI (2.1 g, 13.2 mmol). The mixturewas stirred at 90° C. under a nitrogen atmosphere for 28 hours. Thereaction was then quenched with H₂O and diluted with EtOAc. The organiclayer was separated and washed successively with H₂O and brine, thendried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was purified by flash chromatography (silica, 6:1-1:10hexane:EtOAc) to afford product as a solid (1.12 g, 24%).

The solution of diketone (1.12 g, 3.0 mmol) in conc. HCl (10 mL) anddioxane (10 mL) was heated to reflux overnight. After the solvent wasremoved in vacuo, MeOH (3 mL) was added to dissolve the residue. Ether(300 mL) was then added to precipitate the product (302 mg, 42%) as alight yellow solid.

Example 12

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 27.

To a solution of C₃H₇MgCl (2 M, 50 mmol) in ether (25 mL) at 0° C. wasadded benzaldehyde (5 mL, 42.5 mmol) in ether (50 mL). The resultantsolution was stirred at 0° C. for 30 minutes. The reaction was thenquenched with saturated NH₄Cl and diluted with ether. The organic layerwas separated and washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford thecrude product (7.2 g) which was directly used for the next reactionwithout purification.

To a solution of the above alcohol (7.2 g, 43.9 mmol) and pyridine (7mL, 86.7 mmol) in CH₂Cl₂ (300 mL) at 0° C. was added Dess-Martinperiodinane (19.2 g, 45.3 mmol). The resultant mixture was stirredovernight and quenched with saturated aqueous Na₂S₂O₃ and saturatedaqueous NaHCO₃ (1:1). The organic layer was washed successively with H₂Oand brine, then dried over anhydrous Na₂SO₄, filtered, and concentratedin vacuo. The residue was purified by flash chromatography (silica,8:1-4:1 hexane:EtOAc) to afford product as a colorless oil (6.28 g, 91%for two steps).

To a solution of the above ketone (4.43 g, 27.3 mmol) andN-bromosuccinimde (NBS, 5.5 g, 30.9 mmol) in benzene (150 mL) was added2,2′-azobisisobutyronitrile (AIBN, 0.2 g, 1.2 mmol) at 90° C. Theresultant solution was heated to reflux overnight and then cooled toroom temperature. The brown solution was washed successively with H₂Oand brine, then dried over anhydrous Na₂SO₄, filtered, and concentratedin vacuo. The residue was crystallized from hexanes to afford product asa white solid (6.21 g, 95%).

To a solution of EtONa (2.5 g, 34.9 mmol) in EtOH (200 mL) at 0° C. wasadded diethyl acetamidomalonate (6.7 g, 30.9 mmol) followed by thesolution of the above bromide (6.2 g, 25.8 mmol) in EtOH (100 mL). Theresultant mixture was heated to reflux for 1 h and then quenched withcitric acid (9 g) and diluted with H₂O0 After most solvent was removed,the residue was extracted with EtOAc. The organic layer was washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by flashchromatography (silica, 4:1-2:1 hexane: EtOAc) to afford product as alight yellow solid (8.92 g, 92%).

To a solution of the above ketone (1.4 g, 3.71 mmol) in HOAc (50 mL) wasadded Br₂ (0.7 mL, 13.6 mmol). The mixture was stirred at roomtemperature overnight and then quenched with saturated aqueous NaHCO₃.The mixture was extracted with Et₂O. The organic layer was washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by flashchromatography (silica, 5:1-3:2 hexane:EtOAc) to afford product as ayellow solid (1.23 g, 73%).

To a solution of α-bromo ketone (1.12 g, 2.46 mmol) and Na₂CO₃ (0.4 g,3.77 mmol) in DMSO (30 mL) was added KI (0.45 g, 13.2 mmol). The mixturewas stirred at 90° C. overnight and then quenched with citric acid (2 g)and H₂O (200 mL). The mixture was extracted with EtOAc. The organiclayer was washed successively with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by flash chromatography (silica, 6: 1-1:10 hexane: EtOAc) toafford α-hydroxyl ketone as an oil (0.62 g, 64%).

To a solution of the above alcohol (0.62 g, 1.58 mmol) and pyridine (0.5mL, 6.19 mmol) in CH₂Cl₂ (100 mL) at 0° C. was added Dess-Martinperiodinane (0.9 g, 2.12 mmol). The resultant mixture was stirredovernight and then quenched with saturated aqueous Na₂S₂O₃ and saturatedaqueous NaHCO₃ (1:1). The organic layer was washed successively with H₂Oand brine, then dried over anhydrous Na₂SO₄, filtered, and concentratedin vacuo. The residue was purified by flash chromatography (silica, 9:1-3:2 hexane:EtOAc) to afford product as a yellow oil (287 mg, 30% fortwo steps).

The mixture of the above diketone (272 mg, 0.7 mmol) in conc. HCl (10mL) and dioxane (10 mL) was heated to reflux overnight. After thesolvent was removed in vacuo, MeOH (1 mL) was added to dissolve theresidue. Ether (200 mL) was then added to precipitate the product as ayellow solid (162 mg, 81%).

Example 13

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 28. The compounds were synthesized as presentedin FIG. 28.

Example 14

This example details cloning and expression of a modified polypeptide inE. coli. An introduced translation system that comprises an orthogonaltRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is usedto express the polypeptide containing a non-natural amino acid. The O—RSpreferentially aminoacylates the O-tRNA with a non-natural amino acid.In turn the translation system inserts the non-natural amino acid intothe polypeptide, in response to an encoded selector codon. Amino acidand polynucleotide sequences of O-tRNA and O—RS useful for theincorporation of non-natural amino acids are described in U.S. patentapplication Ser. No. 10/126,927 entitled “In Vivo Incorporation ofUnnatural Amino Acids” and U.S. patent application Ser. No. 10/126,931entitled “Methods and Compositions for the Production of OrthogonaltRNA-Aminoacyl tRNA Synthetase Pairs,” which are incorporated byreference herein. The following O—RS and O-tRNA sequences may also beused:

SEQ ID NO: 1 M. jannaschii mtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 2 HLAD03;an optimized amber tRNA suppressor tRNA SEQ ID NO: 3 HL325A; anoptimized AGGA tRNA frameshift suppressor tRNA SEQ ID NO: 4 AminoacyltRNA synthetase for the RS incorporation of p-azido-L- phenylalaninep-Az-PheRS(6) SEQ ID NO: 5 Aminoacyl tRNA synthetase for the RSincorporation of p-benzoyl-L- phenylalanine p-BpaRS(1) SEQ ID NO: 6Aminoacyl tRNA synthetase for the RS incorporation of propargyl-phenylalanine Propargyl-PheRS SEQ ID NO: 7 Aminoacyl tRNA synthetase forthe RS incorporation of propargyl- phenylalanine Propargyl-PheRS SEQ IDNO: 8 Aminoacyl tRNA synthetase for the RS incorporation of propargyl-phenylalanine Propargyl-PheRS SEQ ID NO: 9 Aminoacyl tRNA synthetase forthe RS incorporation of p-azido- phenylalanine p-Az-PheRS(1) SEQ ID NO:10 Aminoacyl tRNA synthetase for the RS incorporation of p-azido-phenylalanine p-Az-PheRS(3) SEQ ID NO: 11 Aminoacyl tRNA synthetase forthe RS incorporation of p-azido- phenylalanine p-Az-PheRS(4) SEQ ID NO:12 Aminoacyl tRNA synthetase for the RS incorporation of p-azido-phenylalanine p-Az-PheRS(2) SEQ ID NO: 13 Aminoacyl tRNA synthetase forthe RS incorporation of p-azido- phenylalanine (LW1) SEQ ID NO: 14Aminoacyl tRNA synthetase for the RS incorporation of p-azido-phenylalanine (LW5) SEQ ID NO: 15 Aminoacyl tRNA synthetase for the RSincorporation of p-azido- phenylalanine (LW6) SEQ ID NO: 16 AminoacyltRNA synthetase for the RS incorporation of p-azido- phenylalanine(AzPheRS-5) SEQ ID NO: 17 Aminoacyl tRNA synthetase for the RSincorporation of p-azido- phenylalanine (AzPheRS-6)

The transformation of E. coli with plasmids containing the modified geneand the orthogonal aminoacyl tRNA synthetase/tRNA pair (specific for thedesired non-natural amino acid) allows the site-specific incorporationof non-natural amino acid into the polypeptide. The transformed E. coli,grown at 37° C. in media containing between 0.01-100 mM of theparticular non-natural amino acid, expresses modified polypeptide withhigh fidelity and efficiency. The His-tagged polypeptide containing anon-natural amino acid is produced by the E. coli host cells asinclusion bodies or aggregates. The aggregates are solubilized andaffinity purified under denaturing conditions in 6M guanidine HCl.Refolding is performed by dialysis at 4° C. overnight in 50 mM TRIS-HCl,pH8.0, 40CM CuSO₄, and 2% (w/v) Sarkosyl. The material is then dialyzedagainst 20 mM TRIS-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl₂, followed byremoval of the His-tag. See Boissel et al., J. Biol. Chem., (1993)268:15983-93. Methods for purification of polypeptides are well known inthe art and are confirmed by SDS-PAGE, Western Blot analyses, orelectrospray-ionization ion trap mass spectrometry and the like.

Example 15 Testing Non-Natural Amino Acids

This example provides results of four tests that were conducted oncertain illustrative non-natural amino acids as an aid for predictingtheir properties for incorporation into non-natural amino acidpolypeptides.

Oxime Oxime Formation at Stability Intracellular RS Structure pH 6.5 pH4-8 Concentration test

+ *** 600 μM ✓

+++ * 1000-1800 μM

++ ***  60 μM X

++ *** 300 μM

+++ ***  30 μM ✓

++ *** 376 μM

+++ *** Reduced[M + 2]

+++ *** Meta-bolized

+++ ***

+++ **(6.5-7.4) 628 μM(little reduced)

+++

+++ −−

+++ ***

+++ ***

+++ **

++ ** 279 μM

+++ *

+++ **

+++ ***

+++ ***

+++ ***

+++ ***

+++ ***

+++ ***

+++ ***

Example 16 Testing Non-Natural Amino Acids

This example provides the results of pH stability tests that wereconducted on certain illustrative non-natural amino acids as an aid forpredicting their properties for incorporation into non-natural aminoacid polypeptides.

pH 4.0 5.0 6.5 7.4 8.0 1 hour <1 <1 <1 <1 <1 1 day  <1 <1 <1 <1 <1 2days <1 <1 <1 <1 <1 3 days <1 <1 <1 <1 <1 4 days <1 <1 <1 <1 <1 5 days<1 <1 <1 <1 <1 6 days <1 <1 <1 <1 <1 7 days <1 <1 <1 <1 <1 10 days  <1<1 <1 <1 <1

pH 4.0 5.0 6.5 7.4 8.0 1 hour <1 <1 <1 <1 <1 1 day  <1 <1 <1 <1 <1 2days <1 <1 <1 <1 <1 3 days <1 <1 <1 <1 <1 4 days <1 <1 <1 <1 <1 5 days<1 <1 <1 3% <1 6 days 2% <1 <1 4% <1 7 days 2% <1 <1 7% <1 10 days  3%<1 <1 11%  <1

pH 4.0 5.0 6.5 7.4 8.0 1 hour 2% <1 <1 <1 <1 1 day  5% 5% <1 15% 35% 2days 5% 5% <1 15% 38% 3 days 5% 5% <1 15% 40% 4 days 8% 5% <1 15% 45% 5days 8% 5% <1 15% 45% 6 days 8% 5% <1 15% 45% 7 days 9% 5% <1 15% 45% 10days  10%  5% <1 15% 45%

pH 4.0 5.0 6.5 7.4 8.0 1 hour 5% <1 <1 <1  9% 1 day  7% 3% <1 <1 24% 2days 8% 3% <1 <1 30% 3 days 10%  5% 2% <1 30% 4 days 11%  7% 2% <1 30% 5days 11%  7% 2% <1 30% 6 days 11%  7% 2% <1 30% 7 days 11%  8% 2% <1 33%10 days  11%  8% 2% <1 34%

pH 4.0 5.0 6.5 7.4 8.0 1 hour 4.0 5.0 6.5 7.4 8.0 1 day  <1 <1 <1 <1 <12 days <1 <1 <1 <1 <1 3 days <1 <1 <1 <1 <1 4 days <1 <1 <1 <1 <1 5 days<1 <1 <1 <1 <1 6 days <1 <1 <1 <1 <1 7 days <1 <1 <1 <1 <1 10 days  <1<1 <1 <1 <1

pH 4.0 5.0 6.5 7.4 8.0 1 hour 4.0 5.0 6.5 7.4 8.0 1 day  <1 <1 <1 <1 <12 days <1 <1 <1 <1 <1 3 days <1 <1 <1 <1 <1 4 days <1 <1 <1 <1 <1 5 days<1 <1 <1 <1 <1 6 days <1 <1 <1 <1 <1 7 days <1 <1 <1 <1 <1 10 days  <1<1 <1 <1 <1

pH 4.0 5.0 6.5 7.4 8.0 1 hour <1 <1 <1 <1 <1 1 day  <1 <1 <1 <1 <1 2days <1 <1 <1 <1 <1 3 days <1 <1 <1 <1 <1 4 days <1 <1 <1 <1 <1 5 days<1 <1 <1 <1 <1 6 days <1 <1 <1 <1 <1 7 days <1 <1 <1 <1 <1 10 days  <1<1 <1 <1 <1

pH 4.0 5.0 6.5 7.4 8.0 1 hour <1 <1 <1 <1 <1 1 day  <1 <1 <1 <1 <1 2days <1 <1 <1 <1 <1 3 days <1 <1 <1 <1 <1 4 days <1 <1 <1 <1 <1 5 days<1 <1 <1 <1 <1 6 days <1 <1 <1 <1 <1 7 days <1 <1 <1 <1 <1 10 days  <1<1 <1 <1 <1

Example 17

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 29.

To a solution of amino acid pAF (10 g, 41.1 mmol) in H₂O-dioxane (300mL, 1:1) was added NaHCO₃ (12 g, 142.9 mmol) and Boc₂O (12 g, 55.0mmol). The mixture was stirred at room temperature for 7 hours and thenquenched with citric acid. The mixture was extracted with EtOAc. Theorganic layer was washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered and concentrated to afford the N-Boc-pAFas a white solid (13.7 g, quant.).

To NaOH (40 mL, 25% vol.) at 0° C. was added ether (60 mL). A blastshield was placed in front of the reaction flask. To the resultantmixture was added N-nitroso-N-methyl urea (6.0 g, 57.9 mmol) in 3portions over 3 minutes. The reaction was stirred at 0° C. for 10minutes. The diethyl ether and sodium hydroxide layers were then allowedto separate. The organic layer was added to the solution of N-Boc-pAF(5.0 g, 16.2 mmol) in anhydrous THF (20 mL) potionwise (approximately 6additions) over 5 minutes until the starting material had completelydisappeared (monitored by TLC). 5 props of glacial acetic acid were thenadded to quench the reaction. After the organic solvents were removed byrotary evaporation, ethyl acetate was added. The organic layer waswashed successively with saturated NaHCO₃ solution, H₂O and brine, thendried over anhydrous MgSO₄, filtered and concentrated to yield a whitepowder (4.1 g, 80%).

To t-BuOK (60 mL, 1.0 M in THF) was slowly added the solution of theprotected pAF (3.82 g, 11.9 mmol) in freshly distilled methyl propionate(20 mL, 208 mmol). The resultant mixture was stirred at room temperaturefor 30 minutes and quenched with citric acid solution (10%, 300 mL). Themixture was extracted with EtOAc. The organic layer was washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated. The residue was purified by flashchromatography (silica, 4:1-1:1 hexane:EtOAc) to afford product as awhite solid (3.89 g, 87%).

To a solution of the above methyl ester (1.12 g, 2.97 mmol) in dioxane(4 mL) at 0° C. was added LiOH (4 mL, 1 N). The mixture was stirred at0° C. for 3 h and quenched with aqueous citric acid solution (5%, 200mL) and diluted with EtOAc. The organic layer was separated and washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered, and concentrated to afford a white solid (1.02 g, 94%).

To a solution of the above acid (1.0 g, 2.75 mmol) in CH₂Cl₂ (10 mL) at0° C. was added trifluoroacetic acid (10 mL). The mixture was stirred at0° C. for 2 h and then concentrated. To the residue was added MeOH (1mL) followed by HC (1.5 mL, 4 N in dioxane). Ether (200 mL) was thenadded to precipitate the product (701 mg, 96%) as a white solid.

Example 18

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 30.

To t-BuOK (15 mL, 1.0 M in THF) was slowly added the solution of theprotected pAF (1.09 g, 3.4 mmol) in methyl difluoroacetate (6 mL, 68.7mmol). The resultant mixture was stirred at room temperature for 30minutes and quenched with citric acid (5 g, 25.4 mmol) and diluted withH₂O. The mixture was extracted with EtOAc. The organic layer was washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated. The residue was purified by flashchromatography (silica, 20:1-3:2 hexane: EtOAc) to afford product as alight brown solid (1.27 g, 94%).

To a solution of the above methyl ester (1.26 g, 3.17 mmol) in dioxane(30 mL) at 0° C. was added LiOH (30 mL, 1 N). The mixture was stirred at0° C. for 0.5 h and quenched with citric acid (10 g, 51 mmol) anddiluted with H₂O. The mixture was extracted with EtOAc. The organiclayer was washed successively with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered, and concentrated. The residue was purifiedby flash chromatography (silica, 100:1-10:1 CH₂Cl₂: MeOH, 0.5% HOAc) toafford a brown oil (1.19 g, 98%).

To a solution of the above acid (1.19 g, 3.1 mmol) in CH₂Cl₂ (15 mL) at0° C. was added trifluoroacetic acid (15 mL). The mixture was stirredfor 0.5 h and concentrated. To the residue was added MeOH (2 mL)followed by HCl (2 mL, 4 N in dioxane). Ether (200 mL) was then added toprecipitate product (0.82 mg, 82%) as a white solid.

Example 19

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 12 a. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 12 a.

Example 20

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 12 b. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 12 b.

Example 21

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 12 c. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 12 c.

Example 22

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 12 d. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 12 d.

Example 23

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 13. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 13.

Example 24

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 14. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 14.

Example 25

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 15. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 15.

Example 26

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 16 a. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 16 a.

Example 27

This example details the synthesis of the hydroxylamine-containing PEGreagent presented in FIG. 16 b. The hydroxylamine-containing PEG reagentwas produced as described in FIG. 16 b.

Example 28

This example details the synthesis of a hydroxylamine-containing linkerreagent presented in FIG. 18. The hydroxylamine-containing linkerreagent was produced as described in FIG. 18.

Example 29

This example details the synthesis of1,2-bis(4-(bromomethyl)phenyl)disulfane (1) presented in FIG. 36. To anoven-dried roundbottom flask with stirbar under nitrogen gas pressurewas added p-Tolyl disulfide (5.0 g, 20.3 mmol), N-bromo succinimide (8.6g, 48.4 mmol) and 60 mL anhydrous benzene. The solution was heated to 95C. Azobisisobutylnitrile (0.106 g, 0.64 mmol) was added in one portion.The reaction was refluxed for 16 hours. The solvent was removed byrotary evaporation and the brown solid dissolved in 100 mL ethylacetate. The reaction mixture was washed successively with saturatedaqueous sodium bicarbonate solution (2×50 mL), deionized water (1×50 mL)and brine (1×50 mL). The organic layer was separated and dried overanhydrous magnesium sulfate, filtered and concentrated under reducedpressure. The crude product was purified by silica chromatography usinga Biotage Inc. HORIZON™ chromatography system to afford, afterconcentration of the appropriate fractions and removal of traces ofsolvent (vacuum pump), 1,2-bis(4-(bromomethyl)phenyl)disulfane (2.1 g,25%) as a white solid. ¹H NMR spectral data, and a mass spectrum wereobtained. The reaction was repeated to yield (2.0 g, 23%) of theproduct.

Example 30

This example details the synthesis of1,2-bis(4-diethyl-2-acetamidomalonate)phenyldisulfane (2) presented inFIG. 36. To an oven-dried roundbottom flask with stirbar under nitrogengas pressure was added Diethyl acetamidomalonate (6.48 g, 30 mmol) and50 mL anhydrous EtOH. To the solution was added sodium ethoxide (2.6 g,38 mmol) in one portion. The reaction was cooled to 0° C.1,2-bis(4-(bromomethyl)phenyl)disulfane (4.1 g, 10.1 mmol) was dissolvedin 20 mL 1:1 EtOH/THF and added via addition funnel to the cold solutionover the course of 1 hour. The ice-bath was removed and the reactionallowed to stir at room temperature for 6 hours. The solvent was removedby rotary evaporation and the red solid dissolved in 100 mL ethylacetate. The reaction mixture was washed successively with 5% citricacid solution (2×50 mL), deionized water (1×50 mL) and brine (1×50 mL).The organic layer was separated and dried over anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure. The crudeproduct was purified by silica chromatography using a Biotage Inc.HORIZON™ chromatography system to afford, after concentration of theappropriate fractions and removal of traces of solvent (vacuum pump),1,2-bis(4-diethyl-2-acetamidomalonate)phenyldisulfane (5.0 g, 73%) as ayellow solid. ¹H NMR spectral data, a HPLC trace and a mass spectrumwere obtained.

Example 31

This example details the synthesis of 1,2-bis(4-(2-amino-3-propanoicacid)phenyldisulfane (3) presented in FIG. 36. To an oven-driedroundbottom flask with stirbar under nitrogen gas pressure was added1,2-bis(4-diethyl-2-acetamidomalonate)phenyldisulfane (1.0 g, 1.4 mmol),HCl (8 mL, 12 M) and 8 mL 1.4 Dioxane. The reaction was stirred atreflux for 16 hours. The solvent was removed by rotary evaporation andvacuum pump to yield crude 1,2-bis(4-(2-amino-3-propanoicacid)phenyldisulfane (0.75 g, 135%) as a clear oil. ¹H NMR spectral dataand a mass spectrum were obtained.

Example 32

This example details the synthesis ofN,N′-diBoc-1,2-bis(4-(2-amino-3-propanoic acid)phenyldisulfane (4)presented in FIG. 36. To 1,2-bis(4-(2-amino-3-propanoicacid)phenyldisulfane (0.75 g, 1.9 mmol) in a dry roundbottom flask wasadded 5 mL 1,4 dioxane, 5 mL deionized water, Di-t-butyl dicarbonate(0.65 g, 3.0 mmol) and sodium bicarbonate (0.98 g, 12 mmol). Thereaction was stirred at room temperature for 16 hours. The solvent wasremoved by rotary evaporation and the clear oil dissolved in 100 mLethyl acetate. The reaction mixture was washed successively with 5%citric acid solution (5 mL×2), deionized water (50 mL) and brine (50mL). The organic layer was separated and dried over anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure. The crudeproduct was purified by silica chromatography using a Biotage Inc.HORIZON™ chromatography system to afford, after concentration of theappropriate fractions and removal of traces of solvent (vacuum pump),N,N′-diBoc-1,2-bis(4-(2-amino-3-propanoic acid)phenyldisulfane (0.5 g,44% from crude, 52% over 2 steps) as a white solid. ¹H NMR spectraldata, HPLC trace and a mass spectrum were obtained.

Example 33

This example details the synthesis ofN—BOC-2-amino-3-(4-mercaptophenyl)propanoic acid (5) presented in FIG.36. To an oven-dried roundbottom flask with stirbar under nitrogen gaspressure was added N,N′-diBoc-1,2-bis(4-(2-amino-3-propanoicacid)phenyldisulfane (0.5 g, 0.84 mmol), n-Butyl phosphine (0.6 mL, 2.44mmol) and 15 mL anhydrous THF. The reaction was stirred at roomtemperature for 2 hours. The solvent was removed by rotary evaporationand the clear oil dissolved in 50 mL ethyl acetate. The reaction mixturewas washed successively with 5% citric acid solution (2×25 mL),deionized water (25 mL) and brine (25 mL). The organic layer wasseparated and dried over anhydrous magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product was purified bysilica chromatography using a Biotage Inc. HORIZON™ chromatographysystem to afford, after concentration of the appropriate fractions andremoval of traces of solvent (vacuum pump),N—BOC-2-amino-3-(4-mercaptophenyl)propanoic acid (0.5 g, 100%) as awhite solid. ¹H NMR spectral data, HPLC trace and a mass spectrum wereobtained.

Example 34

This example details the synthesis of2-amino-3-(4-mercaptophenyl)propanoic acid hydrochloride (6) presentedin FIG. 36. To an oven-dried roundbottom flask with stirbar was addedN—BOC-2-amino-3-(4-mercaptophenyl)propanoic acid (0.5 g, 1.6 mmol), 10mL anhydrous dichloromethane and 3 mL trifluoroacetic acid. The reactionwas stirred at room temperature for 2 hours. The solvent was removed byrotary evaporation and 1 mL 4.0 M Hydrogen chloride in 1,4-dioxane wasadded. The roundbottom flask was briefly swirled, then 100 mL anhydrousdiethyl ether was added to precipitate2-amino-3-(4-mercaptophenyl)propanoic acid hydrochloride (0.39 g, 100%)was a white solid. ¹H NMR spectral data, HPLC trace and a mass spectrumwere obtained.

Example 35

This example details the synthesis of diethyl2-(4-(2-oxopropylthio)benzyl)-2-acetamidomalonate (7) presented in FIG.37. To an oven-dried roundbottom flask with stirbar under nitrogen gaspressure was added (2) (1.1 g, 1.6 mmol), n-Bu₃P (1.2 mL, 4.8 mmol) and25 mL anhydrous THF (25 mL). The reaction was stirred at roomtemperature for 2 hours. To the reaction was added chloroacetone (0.16mL, 2.0 mmol) and NaHCO₃ (0.98 g, 12 mmol). The reaction was stirred atroom temperature for 2 hours. The solvent was removed by rotaryevaporation and the white solid dissolved in 100 mL ethyl acetate. Thereaction mixture was washed successively with saturated aqueous sodiumbicarbonate solution (50 mL×2), deionized water (50 mL) and brine (50mL). The organic layer was separated and dried over anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure. The crudeproduct was purified by silica chromatography using a Biotage Inc.HORIZON™ chromatography system to afford, after concentration of theappropriate fractions and removal of traces of solvent (vacuum pump),diethyl 2-(4-(2-oxopropylthio)benzyl)-2-acetamidomalonate (0.62 g, 98%)as a white solid. ¹H NMR spectral data, a HPLC trace and a mass spectrumwere obtained.

Example 36

This example details the synthesis of3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid (8) presented inFIG. 37. To an oven-dried roundbottom flask with stirbar under nitrogengas pressure was added 7 (0.62 g, 1.5 mmol), 10 mL 1,4-dioxane and 10 mL12M HCl . The reaction was brought to reflux and allowed to stirovernight. The solvent was removed by rotary evaporation to yield3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid (0.40 g, 99% crude).

Example 37

This example details the synthesis ofN—BOC-3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid (9) presentedin FIG. 37. To 8 (0.35 g, 1.3 mmol) in an oven-dried roundbottom flaskwith stirbar under nitrogen gas pressure was added Di-t-butyldicarbonate (0.63 g, 3.0 mmol), sodium bicarbonate (0.98 g, 12 mmol), 8mL 1,4-dioxane and 8 mL deionized water. The reaction was stirred atroom temperature for 16 hours. The solvent was removed by rotaryevaporation and the clear oil dissolved in 100 mL ethyl acetate. Thereaction mixture was washed successively with 5% citric acid solution(50 mL×2), deionized water (50 mL) and brine (50 mL). The organic layerwas separated and dried over anhydrous magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product was purified bysilica chromatography using a Biotage Inc. HORIZON™ chromatographysystem to afford, after concentration of the appropriate fractions andremoval of traces of solvent (vacuum pump),N—BOC-3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid (0.30 g, 66%from crude) as a white solid. ¹H NMR spectral data, HPLC trace and amass spectrum were obtained.

Example 38

This example details the synthesis ofN—BOC-3-(4-(2-oxopropylsulfinyl)phenyl)-2-aminopropanoic acid (10)presented in FIG. 37. To an oven-dried roundbottom flask with stirbarunder nitrogen gas pressure was added 9 (150 mg, 0.4 mmol), 8 mL glacialacetic acid and 2 mL 30% v/v hydrogen peroxide in water. The reactionwas stirred for 2 hours at room temperature. The solvent was removed byrotary evaporation and the clear oil dissolved in 50 mL ethyl acetate.The reaction mixture was washed successively with 5% citric acidsolution (25 mL×2), deionized water (25 mL) and brine (25 mL). Theorganic layer was separated and dried over anhydrous magnesium sulfate,filtered and concentrated under reduced pressure. The crude product waspurified by silica chromatography using a Biotage Inc. HORIZON™chromatography system to afford, after concentration of the appropriatefractions and removal of traces of solvent (vacuum pump),N—BOC-3-(4-(2-oxopropylsulfinyl)phenyl)-2-aminopropanoic acid (0.13 g,86% crude). A HPLC trace and a mass spectrum were obtained.

Example 39

This example details the synthesis of3-(4-(2-oxopropylsulfinyl)phenyl)-2-aminopropanoic acid (11) presentedin FIG. 37. To an oven-dried roundbottom flask with stirbar was addedN—BOC-3-(4-(2-oxopropylsulfinyl)phenyl)-2-aminopropanoic acid 10 (0.13g, 0.35 mmol), 10 mL anhydrous dichloromethane and 3 mL trifluoroaceticacid. The reaction was stirred at room temperature for 2 hours. Thesolvent was removed by rotary evaporation and 1 mL 4.0 M Hydrogenchloride in 1,4-dioxane was added. The roundbottom flask was brieflyswirled, then 100 mL anhydrous diethyl ether was added to precipitate3-(4-(2-oxopropylsulfinyl)phenyl)-2-aminopropanoic acid (0.072 g, 74%from crude) as a white solid. ¹H NMR spectral data, HPLC trace and amass spectrum were obtained.

Example 40

This example details the synthesis ofN—BOC-3-(4-(2-oxopropylsulfonyl)phenyl)-2-aminopropanoic acid (12)presented in FIG. 37. To an oven-dried roundbottom flask with stirbarunder nitrogen gas pressure was added 9 (150 mg, 0.4 mmol), 8 mL glacialacetic acid and 2 mL 30% v/v hydrogen peroxide in water. The reactionwas stirred at room temperature for 24 hours. The solvent was removed byrotary evaporation and the clear oil dissolved in 50 mL ethyl acetate.The reaction mixture was washed successively with 5% citric acidsolution (25 mL×2), deionized water (25 mL) and brine (25 mL). Theorganic layer was separated and dried over anhydrous magnesium sulfate,filtered and concentrated under reduced pressure. The crude product waspurified by silica chromatography using a Biotage Inc. HORIZON™chromatography system to afford, after concentration of the appropriatefractions and removal of traces of solvent (vacuum pump),N—BOC-3-(4-(2-oxopropylsulfonyl)phenyl)-2-aminopropanoic acid (12) (0.13g, 86% crude). A HPLC trace and a mass spectrum were obtained.

Example 41

This example details the synthesis of3-(4-(2-oxopropylsulfonyl)phenyl)-2-aminopropanoic acid (13) presentedin FIG. 37. To an oven-dried roundbottom flask with stirbar was addedN—BOC-3-(4-(2-oxopropylsulfonyl)phenyl)-2-aminopropanoic acid 12 (0.13g, 0.35 mmol), 10 mL anhydrous dichloromethane and 3 mL trifluoroaceticacid. The reaction was stirred at room temperature for 2 hours. Thesolvent was removed by rotary evaporation and 1 mL 4.0 M Hydrogenchloride in 1,4-dioxane was added. The roundbottom flask was brieflyswirled, then 100 mL anhydrous diethyl ether was added to precipitate3-(4-(2-oxopropylsulfonyl)phenyl)-2-aminopropanoic acid (0.067 g, 65%from crude) as a white solid. ¹H NMR spectral data, HPLC trace and amass spectrum were obtained.

Example 42

This example details the synthesis of3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid (14) presented inFIG. 37. To an oven-dried roundbottom flask with stirbar was addedN—BOC-3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid 9 (0.10 g,0.28 mmol), 10 mL anhydrous dichloromethane and 3 mL trifluoroaceticacid. The reaction was stirred at room temperature for 2 hours. Thesolvent was removed by rotary evaporation and 1 mL 4.0 M Hydrogenchloride in 1,4-dioxane was added. The roundbottom flask was brieflyswirled, then 100 mL anhydrous diethyl ether was added to precipitate3-(4-(2-oxopropylthio)phenyl)-2-aminopropanoic acid (0.062 g, 85% fromcrude) as a white solid. ¹H NMR spectral data, HPLC trace and a massspectrum were obtained.

Example 43

This example details the synthesis ofN—BOC-3-(4-(2-oxocyclopentylthio)phenyl)-2-aminopropanoic acid (15)presented in FIG. 38. To an oven-dried roundbottom flask with stirbarunder nitrogen gas pressure was added 5 (0.15 g, 0.76 mmol),2-chlorocyclopentanone (0.12 mL, 1.25 mmol), sodium bicarbonate (0.98 g,12 mmol), 15 mL anhydrous THF. The reaction was stirred at roomtemperature for 16 hours. The solvent was removed by rotary evaporationand the white solid dissolved in 100 mL ethyl acetate. The reactionmixture was washed successively with saturated aqueous sodiumbicarbonate solution (50 mL×2), deionized water (50 mL) and brine (50mL). The organic layer was separated and dried over anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure. The crudeproduct was purified by silica chromatography using a Biotage Inc.HORIZON™ chromatography system to afford, after concentration of theappropriate fractions and removal of traces of solvent (vacuum pump),N—BOC-3-(4-(2-oxocyclopentylthio)phenyl)-2-aminopropanoic acid (0.15 g,51%) as a white solid. A HPLC trace and a mass spectrum were obtained.

Example 44

This example details the synthesis of3-(4-(2-oxocyclopentylthio)phenyl)-2-aminopropanoic acid (16) presentedin FIG. 38. To an oven-dried roundbottom flask with stirbar was addedN—BOC-3-(4-(2-oxocyclopentylthio)phenyl)-2-aminopropanoic acid 15 (0.15g, 0.39 mmol), 10 mL anhydrous dichloromethane and 3 mL trifluoroaceticacid. The reaction was stirred at room temperature for 2 hours. Thesolvent was removed by rotary evaporation and 1 mL 4.0 M Hydrogenchloride in 1,4-dioxane was added. The roundbottom flask was brieflyswirled, then 100 mL anhydrous diethyl ether was added to precipitate3-(4-(2-oxocyclopentylthio)phenyl)-2-aminopropanoic acid (0.108 g, 100%)as a white solid. ¹H NMR spectral data, HPLC trace and a mass spectrumwere obtained.

Example 45

This example details the synthesis ofN—BOC-3-(4-(2-oxobutylthio)phenyl)-2-aminopropanoic acid (18) presentedin FIG. 38. To an oven-dried roundbottom flask with stirbar undernitrogen gas pressure was added 5 (0.15 g, 0.76 mmol),1-bromo-2-butanone (0.12 mL, 1.25 mmol), sodium bicarbonate (0.98 g, 12mmol), 15 mL anhydrous THF. The reaction was stirred at room temperaturefor 16 hours. The solvent was removed by rotary evaporation and thewhite solid dissolved in 100 mL ethyl acetate. The reaction mixture waswashed successively with saturated aqueous sodium bicarbonate solution(50 mL×2), deionized water (50 mL) and brine (50 mL). The organic layerwas separated and dried over anhydrous magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product was purified bysilica chromatography using a Biotage Inc. HORIZON™ chromatographysystem to afford, after concentration of the appropriate fractions andremoval of traces of solvent (vacuum pump),N—BOC-3-(4-(2-oxobutylthio)phenyl)-2-aminopropanoic acid (0.15 g, 51%)as a white solid. A HPLC trace and a mass spectrum were obtained.

Example 46

This example details the synthesis of Compounds 19-22 presented in FIG.38. Compounds 19-22 are synthesized using methodology analogous to thatdescribed for Compounds 10-14.

Example 47

This example details the synthesis of3-(4-(2-oxobutylthio)phenyl)-2-aminopropanoic acid (23) presented inFIG. 38. To an oven-dried roundbottom flask with stirbar was addedN—BOC-3-(4-(2-oxobutylthio)phenyl)-2-aminopropanoic acid 18 (0.15 g,0.56 mmol), 10 mL anhydrous dichloromethane and 3 mL trifluoroaceticacid. The reaction was stirred at room temperature for 2 hours. Thesolvent was removed by rotary evaporation and 1 mL 4.0 M Hydrogenchloride in 1,4-dioxane was added. The roundbottom flask was brieflyswirled, then 100 mL anhydrous diethyl ether was added to precipitate3-(4-(2-oxobutylthio)phenyl)-2-aminopropanoic acid (0.149 g, 100%) as awhite solid. ¹H NMR spectral data, HPLC trace and a mass spectrum wereobtained.

Example 48

This example details the synthesis of Compounds 24-27 presented in FIG.39. Compounds 24-27 are synthesized using methodology analogous to thatdescribed for Compounds 10-14.

Example 49

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 31.

To t-BuOK (15 mL, 1.0 M in THF) is slowly added the solution of theprotected pAF (1.0 g, 3.1 mmol) in methyl trifluoroacetate (5 mL, 50mmol). The reaction mixture is stirred at room temperature for 30minutes and quenched with citric acid (5 g, 25.4 mmol) and diluted withEtOAc. The organic layer is washed successively with H₂O and brine, thendried over anhydrous Na₂SO₄, filtered and concentrated. The residue ispurified by flash chromatography (silica, 20:1-3:2 hexane:EtOAc) toafford product (1.07 g, 83%) as a light brown solid.

To a solution of the above methyl ester (1.0 g, 2.4 mmol) in dioxane (30mL) at 0° C. is added LiOH (30 mL, 1 N). The mixture is stirred at 0° C.for 0.5 h and quenched with citric acid (10 g, 51 mmol) and diluted withH₂O. The mixture is extracted with EtOAc. The organic layer is washedsuccessively with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue is purified by flashchromatography (silica, 100:1-10:1 CH₂Cl₂: MeOH, 0.5% HOAc) to afford abrown oil (0.87 g, 98%).

To a solution of the above acid (1.0 g, 2.5 mmol) in CH₂Cl₂ (15 mL) at0° C. is added trifluoroacetic acid (15 mL). The resultant mixture isstirred for 0.5 h and concentrated in vacuo. To the residue was addedMeOH (2 mL) followed by HC (2 mL, 4 N in dioxane). Ether (200 mL) wasthen added to precipitate product (0.56 g, 75%) as a white solid.

Example 50

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 32.

To t-BuOK (15 mL, 1.0 M in THF) is slowly added the solution of theprotected pAF (1.0 g, 3.1 mmol) in methyl pentafluoropropionate (8 mL,62 mmol). The resultant mixture is stirred at room temperature for 1 hand quenched with citric acid (5 g, 25.4 mmol) and diluted with H₂O (100mL). After most solvent is removed, the residue is extracted with EtOAc.The organic layer is washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered and concentrated. The residue ispurified by flash chromatography (silica, 20:1-3:2 hexane:EtOAc) toafford product (1.1 g, 76%) as a light brown solid.

To a solution of the above methyl ester (1.0 g, 2.1 mmol) in dioxane (30mL) at 0° C. is added LiOH (30 mL, 1 N). The resultant mixture isstirred at 0° C. for 0.5 h and quenched with citric acid (10 g, 51 mmol)and H₂O. The mixture is extracted with EtOAc. The organic layer iswashed successively with H₂O and brine, then dried over anhydrousNa₂SO₄, filtered and concentrated. The residue is purified by flashchromatography (silica, 100:1-10:1 CH₂Cl₂: MeOH, 0.5% HOAc) to affordproduct (0.8 g, 84%) as a yellow solid.

To a solution of the above acid (0.7 g, 2.5 mmol) in CH₂Cl₂ (15 mL) at0° C. is added trifluoroacetic acid (15 mL). The mixture is stirred atthe same temperature for 0.5 h and concentrated. To the residue is addedMeOH (2 mL) followed by HC (2 mL, 4 N in dioxane). Ether (200 mL) isthen added to precipitate the product (0.62 g, 70%) as a white solid.

Example 51

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 33.

To a stirred solution of alcohol 1 (2.35 g, 7.6 mmol) and pyridine (1.5mL, 18.6 mmol) in CH₂Cl₂ (150 mL) at 0° C. was added Dess-Martinperiodinane (3.5 g, 8.3 mmol). The mixture was stirred at roomtemperature overnight and quenched with saturated aqueous Na₂S₂O₃—NaHCO₃(1:1, 100 mL) and diluted with CH₂Cl₂. The organic layer was separatedand washed with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo. Purification of the residue by flashchromatography (silica, 20:1-3:1 hexane:EtOAc) to afford aldehyde 2 as awhite solid (2.15 g, 92%). ESI-MS, m/z: 292 (M⁺−OH), 232, 204, 175, 131,115 (100).

To a stirred solution of diisopropylamine (0.33 mL, 2.33 mmol) in THF(60 mL) at 0° C. was added n-butyllithium (1.46 mL, 2.34 mmol). Themixture was stirred at 0° C. for 20 minutes and cooled to −78° C. andcyclopentanone was then added. After the mixture was stirred for 20minutes at −78° C., the solution of aldehyde 2 (0.5 g, 1.63 mmol) in THF(20 mL, washed with 20 mL) was added. The resultant mixture was stirredat −78° C. for 1.0 h and quenched with saturated aqueous NH₄Cl solution.After most of the solvent was removed, the residue was extracted withEtOAc. The organic layer was washed with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 10:1-1:1 hexane:EtOAc)afforded 3 as a colorless oil (482 mg, 80%).

To a stirred solution of alcohol 3 (0.44 g, 1.13 mmol) and pyridine (0.6mL, 7.44 mmol) in CH₂Cl₂ (150 mL) at 0° C. was added Dess-Martinperiodinane (0.6 g, 1.41 mmol). The mixture was stirred at roomtemperature overnight. The reaction was quenched with saturated aqueousNa₂S₂O₃—NaHCO₃ (1:1, 100 mL) and extracted with CH₂Cl₂. The organiclayers were combined and washed with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 20:1-2:1 hexane:EtOAc) gavethe diketone 4 (342 mg, 78%) as a colorless oil. ESI-MS, m/z: 412(M⁺+Na), 356, 312, 230, 212, 184, 146 (100).

To a solution of methyl ester 4 (330 mg, 0.85 mmol) in dioxane (4 mL) at0° C. was added LiOH (4 mL, 1 N). The resultant mixture was stirred at0° C. for 30 minutes and quenched with aqueous citric acid solution (5%,100 mL). The mixture was extracted with EtOAc. The organic layer waswashed successively with H₂O and brine, then dried over anhydrousNa₂SO₄, filtered and concentrated to afford a white solid (310 mg, 97%).ESI-MS, m/z: 342, 330 (M⁺−COOH), 298, 230, 185, 119 (100).

To a solution of acid 5 (310 mg, 0.83 mmol) in CH₂Cl₂ (4 mL) at 0° C.was added trifluoroacetic acid (4 mL). The mixture was stirred at 0° C.for 30 minutes and concentrated in vacuo. To the residue was added MeOH(1 mL) followed by HCl (1 mL, 4 N in dioxane). Ether (100 mL) was thenadded to precipitate product (241 mg, 94%) as a white solid. ESI-MS,m/z: 298 (M⁺+Na), 276 (M⁺+1), 230 (M⁺−COOH), 184, 119 (100).

Example 52

This example details the synthesis of the dicarbonyl-containing aminoacid presented in FIG. 34.

To a stirred solution of alcohol (6.0 g, 19.4 mmol) and pyridine (12 mL,150 mmol) in CH₂Cl₂ (400 mL) at 0° C. was added Dess-Martin periodinane(14.2 g, 33.4 mmol). The mixture was stirred at room temperatureovernight. The reaction was quenched with saturated aqueousNa₂S₂O₃—NaHCO₃ (1:1, 300 mL) and extracted with CH₂Cl₂. The organiclayers were combined and washed with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 1:100-1:1 hexane:EtOAc)afforded the aldehyde (5.48 g, 92%) as a white solid.

To a solution of the above aldehyde (3.41 g, 11.1 mmol) in acetone (70mL) was added KMnO₄ (2.5 g, 15.8 mmol) in H₂O (10 mL). The resultantmixture was stirred at room temperature overnight. After most solventwas removed, the residue was dissolved in citric acid aqueous solution(5%, 300 mL) and extracted with EtOAc. The organic layers were combinedand washed with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo to afford product as a white solid(2.83 g, 79%) which was directly used for the next step without furtherpurification.

To a solution of the above acid (2.83 g, 8.76 mmol) in DMF (60 mL) at 0°C. were added 1-amino-2-propanol (1.4 mL, 17.9 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 4.1 g,21.4 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 2.2 g, 18.5 mmol) andN N-diisopropylethylamine (DIEA, 9 mL, 51.6 mmol). The mixture wasstirred at room temperature overnight and then quenched with citric acidaqueous solution (5%, 200 mL) and extracted with EtOAc. The organiclayers were combined and washed with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 10:1-1:1 hexane:EtOAc)afforded product (2.45 g, 74%) as a white foam.

To a stirred solution of the above alcohol (2.44 g, 6.4 mmol) andpyridine (4 mL, 49.6 mmol) in CH₂Cl₂ (100 mL) at 0° C. was addedDess-Martin periodinane (4.1 g, 9.7 mmol). The mixture was stirred atroom temperature overnight. The reaction was quenched with saturatedaqueous Na₂S₂O₃—NaHCO₃ (1:1, 300 mL) and extracted with CH₂Cl₂. Theorganic layer was washed with H₂O and brine, then dried over anhydrousNa₂SO₄, filtered and concentrated in vacuo. Purification of the residueby flash chromatography (silica, 1:1-1:3 hexane:EtOAc) afforded product(1.84 g, 76%) as a yellow solid.

To a solution of the above methyl ester (1.72 g, 4.6 mmol) in dioxane(10 mL) at 0° C. was added LiOH (10 mL, 1 N). The mixture was stirred atthe same temperature for 3 h and quenched with citric acid aqueoussolution (5%). The mixture was extracted with EtOAc. The organic layerwas washed successively with H₂O and brine, then dried over anhydrousNa₂SO₄, filtered, and concentrated to afford product (1.7 g) as a solidwhich was used directly for the next step without purification.

To a solution of the above acid (1.7 g, 4.7 mmol) in CH₂Cl₂ (15 mL) at0° C. was added trifluoroacetic acid (15 mL). The mixture was stirred atthe same temperature for 2 h and concentrated in vacuo. To the residuewas added HCl (1.5 mL, 4 N in dioxane) followed by ether (400 mL). Theprecipitated product (1.52 g, 90% for 2 steps) was collected as a whitesolid.

Example 53

This example details the synthesis of the hydrazide-containing aminoacid presented in FIG. 44.

To a solution of aldehyde (410 mg, 1.34 mmol) in EtOH (15 mL) was addedformic hydrazide (170 mg, 2.83 mmol). The reaction mixture was stirredat room temperature for 1 hour. After most solvent was removed, theresidue was extracted with CH₂Cl₂. The organic layers were combined andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 1:6-1:1 hexane:EtOAc) afforded a white solid(390 mg, 83%).

To a solution of the above methyl ester (349 mg, 1 mmol) in dioxane (7mL) was added LiOH (7 mL, 1 N) at 0° C. The mixture was stirred at thesame temperature for 10 minutes and quenched with citric acid (2.5 g)and diluted with H₂O. The mixture was extracted with EtOAc. The organiclayer was washed successively with H₂O and brine, then dried overanhydrous Na₂SO₄, filtered, and concentrated to give a white solid (290mg, 87%).

To a solution of the above acid (290 mg, 0.87 mmol) in CH₂Cl₂ (20 mL) at0° C. was added trifluoroacetic acid (20 mL). The mixture was stirredfor 20 minutes and concentrated. To the residue was added MeOH (1 mL)followed by HC (1.0 mL, 4 N in dioxane). Ether (200 mL) was then addedto precipitate product (195 mg, 83%) as a light yellow solid.

Example 54

This example details the synthesis of the hydrazide-containing aminoacid presented in FIG. 45.

To a solution of N-Boc-4-hydroxymethylphenylalanine (11.73 g, 39.8 mmol)in DMF (100 mL) was added alanine methyl ester hydrochloride (9.0 g,64.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC, 15.4 g, 80.3 mmol), N,N-diisopropylethylamine (DIEA, 30 mL, 172mmol) and 1-hydroxybenzotriazole hydrate (HOBt, 8.4 g, 70.6 mmol). Thereaction mixture was stirred at room temperature overnight and thendiluted with EtOAc. The organic layer was separated and washedsuccessively with H₂O, citric acid (5%), H₂O, NaHCO₃, H₂O and brine,then dried over anhydrous Na₂SO₄, filtered, and concentrated to affordthe protected dipeptide as a white solid (13.74 g, 91%).

To a solution of the above protected dipeptide (10.33 g, 27.2 mmol) inCH₂Cl₂ (300 mL) at 0° C. were added pyridine (8 mL, 99.1 mmol) andDess-Martin periodinane (14 g, 33.0 mmol). The reaction mixture wasstirred overnight and then quenched with saturated aqueousNaHCO₃/Na₂S203 (1:1). The organic layer was washed successively withH₂O, citric acid, H₂O and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated. The residue was purified by flashchromatography (silica, 9:1-1:1 hexane:EtOAc) to afford product (10.12g, 98%) as a white solid.

To a solution of dipeptide aldehyde (10.1 g, 26.7 mmol) in EtOH (200 mL)was added acetic hydrazide (3.7 g, 45 mmol). The reaction mixture wasstirred at room temperature for 30 minutes and concentrated. To theresidue were added H₂O (1 L) and CH₂C12 (500 mL). The organic layer wasseparated and concentrated to afford a white solid (11.21 g, 97%).

To a solution of the above methyl ester (11.1 g, 25.6 mmol) in dioxane(50 mL) at 0° C. was added LiOH (50 mL, 1 N). The mixture was stirred atthe same temperature for 30 minutes and then quenched with citric acid(20 g) and diluted with H2O (200 mL). The mixture was extracted withEtOAc. The organic layer was washed successively with H2O and brine,then dried over anhydrous Na2SO4, filtered, and concentrated to afford awhite solid (9.52 g, 88%).

To a solution of the above acid (9.5 g, 22.6 mmol) in CH2C12 (50 mL) at0° C. was added trifluoroacetic acid (50 mL). The mixture was stirred at0° C. for 1 h and concentrated in vacuo. To the residue was added HCl (7mL, 4 N in dioxane) followed by ether (500 mL). The precipitate wascollected as a white solid (7.25 g, 90%).

Example 55

This example details the synthesis of the oxime-containing amino acidpresented in FIG. 46A.

To a solution of the aldehyde (3.0 g) in MeOH/H+ was added 2 equivalentsof hydroxylamine hydrochloride. The reaction mixture was stirred at roomtemperature for 2 hours and concentrated. To the residue was added H₂O(200 mL) followed by CH₂Cl₂. The organic layer was separated andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 3:7-1:9 hexane:EtOAc) yielded the product (96%)as a solid.

To a solution of the above methyl ester (3.0 g) in dioxane (10 mL) at 0°C. was added LiOH (10 mL, 1 N). The mixture was stirred at the sametemperature for 3 hours and then quenched by the addition of citric acid(5 g) and diluted with H₂O. The mixture was extracted with EtOAc. Theorganic layer was washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered, and concentrated to afford a solid.Then, to a solution of the resulting acid in CH₂Cl₂ (20 mL) at 0° C. wasadded trifluoroacetic acid (20 mL). The reaction mixture was stirred at0° C. for 1 hour and concentrated. To the residue was added MeOH (1 mL)followed by the addition of HCl (2.0 mL, 4 N in dioxane). Ether (200 mL)was then added to precipitate the product (87%) as a solid.

Example 56

This example details the synthesis of the oxime-containing amino acidpresented in FIG. 46B.

To a solution of the aldehyde (3.0 g) in MeOH/H⁺ was added 2 equivalentsof methoxyamine hydrochloride. The reaction mixture was stirred at roomtemperature for 2 hours and concentrated. To the residue was added H₂O(200 mL) followed by CH₂Cl₂. The organic layer was separated andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 3:7-1:9 hexane:EtOAc) yielded the product (93%)as a solid.

To a solution of the above methyl ester (3.0 g) in dioxane (10 mL) at 0°C. was added LiOH (10 mL, 1 N). The mixture was stirred at the sametemperature for 3 hours and then quenched by the addition of citric acid(5 g) and diluted with H₂O. The mixture was extracted with EtOAc. Theorganic layer was washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered, and concentrated to afford a solid.Then, to a solution of the resulting acid in CH₂Cl₂ (20 mL) at 0° C. wasadded trifluoroacetic acid (20 mL). The reaction mixture was stirred at0° C. for 1 hour and concentrated. To the residue was added MeOH (1 mL)followed by the addition of HCl (2.0 mL, 4 N in dioxane). Ether (200 mL)was then added to precipitate the product (89%) as a solid.

Example 57

This example details the synthesis of the hydrazine-containing aminoacid presented in FIG. 46C.

To a solution of the aldehyde (3.0 g) in MeOH/H⁺ was added 2 equivalentsof methylhydrazine. The reaction mixture was stirred at room temperaturefor 2 hours and concentrated. To the residue was added H₂O (200 mL)followed by CH₂Cl₂. The organic layer was separated and concentrated invacuo. Purification of the residue by flash chromatography (silica,3:7-1:9 hexane:EtOAc) yielded the product (93%) as a solid.

To a solution of the above methyl ester (3.0 g) in dioxane (10 mL) at 0°C. was added LiOH (10 mL, 1 N). The mixture was stirred at the sametemperature for 3 hours and then quenched by the addition of citric acid(5 g) and diluted with H₂O. The mixture was extracted with EtOAc. Theorganic layer was washed successively with H₂O and brine, then driedover anhydrous Na₂SO₄, filtered, and concentrated to afford a solid.Then, to a solution of the resulting acid in CH₂Cl₂ (20 mL) at 0° C. wasadded trifluoroacetic acid (20 mL). The reaction mixture was stirred at0° C. for 1 hour and concentrated. To the residue was added MeOH (1 mL)followed by the addition of HCl (2.0 mL, 4 N in dioxane). Ether (200 mL)was then added to precipitate the product (89%) as a solid.

Example 58

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 48A.

To a solution of mPEG(30K)-OH (1.0 g, 0.033 mmol) in anhydrous CH₂Cl₂(10 mL) was added p-nitrophenol chloroformate (60 mg, 0.28 mmol). Themixture was stirred at room temperature for 15 hours. Ether (200 mL) wasadded. The precipitate was filtered, washed with ether and dried invacuo to afford product (1.0 g, 100%) as a white powder.

To a solution of t-butyl 3-hydroxyethylcarbamate (1.75 g, 10 mmol) inTHF (60 mL) were added N-hydroxyphthalimide (3.2 g, 20 mmol),triphenylphosphine (2.0 g, 15 mmol). The reaction mixture was stirred atroom temperature for 10 minutes and then cooled to 0° C.Diisopropylazodicarboxylate (DIAD, 2.0 mL, 10.5 mmol) was added dropwisevia syringe over 1 hour. The icebath was removed and the mixture wasstirred overnight and concentrated. The white solid dissolved in ethylacetate (100 mL). The reaction mixture was washed successively withsaturated aqueous sodium bicarbonate solution (100 mL), H₂O (100 mL) andbrine (100 mL), then dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. The crude product was purified by flashchromatography (silica, 100:1-10:1 hexane:EtOAc) to afford the titlecompound (2.6 g, 81%) as a white solid.

To a solution of the Boc-protected linker (2.0 g, 9.1 mmol) in CH₂Cl₂ (5mL) was added trifluoroacetic acid (5 mL). The resultant mixture wasstirred at room temperature for 1 hour and concentrated. To the residuewas added HCl (4 N in dioxane, 1.5 mL) followed by the addition of Et₂O(150 mL). The precipitate was filtered, washed with ether and dried invacuo to afford the amine linker (1.1 g, 85%) as a white solid.

To a mixture of mPEG(30 K) p-nitrophenolcarbonate (1.0 g, 0.033 mmol)and amine linker (53 mg, 0.21 mmol) in DMF-CH₂Cl₂ (10 mL, 1:2) wereadded diisopropylethylamine (50 μL, 0.28 mmol) and DMAP (5 mg, 0.041mmol). The resultant mixture was stirred at room temperature for 15hours. Ether (200 mL) was added. The precipitate was filtered, washedwith ether and dried in vacuo to afford product (0.83 g, 83%) as a whitepowder.

To a solution of mPEG phthalimide (30K, 0.8 g, 0.0266 mmol) in MeOH (5mL) was added hydrazine (8.5 μL, 0.27 mmol). The resultant mixture wasstirred at 45° C. for 1.0 hours. After the reaction was cooled to roomtemperature, CH₂Cl₂ (150 mL) was added and the solution was washed withaqueous HCl solution (0.1 N, 100 mL). The aqueous layer was extractedwith CH₂Cl₂ (150 mL). The organic layers were combined and washed withH₂O (100 mL), then dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was dissolved in CH₂Cl₂ (5 mL). Ether (200 mL)was added to precipitate the hydroxylamine product (0.72 g, 90%) as awhite powder.

Example 59

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 48B.

To a solution of mPEG(30K)-OH (1.0 g, 0.033 mmol) in anhydrous CH₂Cl₂(10 mL) was added p-nitrophenol chloro formate (60 mg, 0.28 mmol). Themixture was stirred at room temperature for 15 hours. Ether (200 mL) wasadded. The precipitate was filtered, washed with ether and dried invacuo to afford product (1.0 g, 100%) as a white powder.

To a solution of t-butyl 2-hydroxyethylcarbamate (2.8 mL, 18 mmol) inTHF (60 mL) were added N-hydroxyphthalimide (5.8 g, 36 mmol),triphenylphosphine (3.6 g, 27 mmol). The reaction mixture was stirred atroom temperature for 10 minutes and then cooled to 0° C.Diisopropylazodicarboxylate (DIAD, 3.6 mL, 19 mmols) was added dropwisevia syringe over 1 hour. The icebath was removed and the mixture wasstirred overnight and concentrated. The white solid dissolved in ethylacetate (100 mL). The reaction mixture was washed successively withsaturated aqueous sodium bicarbonate solution (2×50 mL), H₂O (50 mL) andbrine (50 mL), then dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. The crude product was purified by flashchromatography using a Biotage Inc. HORIZON™ chromatography system toafford the title compound with impurities (12 g, 206%) as a white solid.

To a solution of the crude Boc-protected linker (12 g) in CH₂Cl₂ (5 mL)was added trifluoroacetic acid (5 mL). The resultant mixture was stirredat room temperature for 1 hour and concentrated. To the residue wasadded HCl (4 N in dioxane, 1.5 mL) followed by the addition of Et₂O (150mL). The precipitate was filtered, washed with ether and dried in vacuoto afford the amine linker (3.0 g, 68% for two steps) as a white solid.

To a mixture of mPEG(30 K) p-nitrophenolcarbonate (1.0 g, 0.033 mmol)and amine linker (50 mg, 0.21 mmol) in DMF-CH₂Cl₂ (10 mL, 1:2) wereadded diisopropylethylamine (50 μL, 0.28 mmol) and4-dimethylaminopyridine (4 mg, 0.033 mmol). The resultant mixture wasstirred at room temperature for 15 hours. Ether (200 mL) was added. Theprecipitate was filtered, washed with ether and dried in vacuo to affordproduct (0.81 g, 81%) as a white powder.

To a solution of mPEG(30K) phthalimide (0.8 g, 0.0266 mmol) in MeOH (5mL) was added hydrazine (8.5 μL, 0.27 mmol). The resultant mixture wasstirred at 45° C. for 1.0 hour. After the reaction was cooled to roomtemperature, CH₂Cl₂ (150 mL) was added and the solution was washed withaqueous HCl solution (0.1 N, 100 mL). The aqueous layer was extractedwith CH₂Cl₂ (150 mL). The organic layers were combined and washed withH₂O (100 mL), then dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was dissolved in CH₂Cl₂ (5 mL). Ether (200 mL)was added to precipitate the hydroxylamine product (0.68 g, 85%) as awhite powder.

Example 60

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 49A.

To a solution of N-(3-bromopropyl)phthalimide (4.0 g, 15.0 mmol) in DMF(50 mL) at 0° C. were added K₂CO₃ (10 g, 73 mmol) and t-butylN-hydroxycarbamate (2.5 g, 18.8 mmol). The reaction mixture was stirredat room temperature for 3 hours. The mixture was diluted with H₂O (200mL) and extracted with EtOAc (200 mL). The organic layer was washed withH₂O and brine, then dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 20:1-3:1 hexane:EtOAc) afforded product (3.5 g,72%) as a colorless oil

To a solution of the above phthalimide (500 mg, 1.6 mmol) in EtOH (10mL) was added hydrazine (0.25 mL, 8.0 mmol). The resultant mixture wasstirred at room temperature for 3 hours. After removal of theprecipitate, the filtrate was concentrated. The residue was left in highvacuum overnight. Purification of the residue by flash chromatography(silica, 3: 1-1:1 EtOAc:MeOH) afforded the amine linker (252 mg, 85%) asa white solid.

To a solution of mPEG(30K)-OH (1.0 g, 0.033 mmol) in anhydrous CH₂Cl₂(10 mL) was added p-nitrophenol chloroformate (60 mg, 0.28 mmol). Themixture was stirred at room temperature for 15 hours. Ether (200 mL) wasadded. The precipitate was filtered, washed with ether and dried invacuo to afford product (1.0 g, 100%) as a white solid.

To a solution of the above activated mPEG(30K) (3.0 g, 0.1 mmol) inanhydrous CH₂Cl₂ (30 mL) were added diisopropylethylamine (88 μL, 0.5mmol) and the amine linker (76 mg, 0.4 mmol). The resultant mixture wasstirred at room temperature for 15 hours. Ether (700 mL) was added tothe reaction mixture. The precipitate was filtered, washed with etherand dried in vacuo to afford a white powder (2.8 g, 93%).

To a solution of Boc protected mPEG(30 K) (2.0 g, 0.067 mmol) inanhydrous CH₂Cl₂ (10 mL) at 0° C. was added trifluoroacetic acid (10mL). The resultant mixture was stirred at room temperature for 5 hours.Ether (500 mL) was added to the reaction mixture. The precipitate wasfiltered, washed with ether and dried in vacuo to afford a white powder(1.8 g, 90%).

Example 61

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 49B.

To a solution of t-butyl N-hydroxycarbamate (5.0 g, 37.6 mmol) in DMF(30 mL) at 0° C. were added K₂CO₃ (12 g, 87.6 mmol) andN-(2-bromoethyl)phthalimide (10.0 g, 39.7 mmol). The reaction mixturewas stirred at room temperature for 3 hours. The mixture was dilutedwith H₂O (200 mL) and extracted with EtOAc (200 mL). The organic layerwas washed with H₂O and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo. Purification of the residue by flashchromatography (silica, 20:1-1:1 hexane:EtOAc) afforded product (5.2 g,55%) as a white solid.

To a solution of the above phthalimide (500 mg, 1.6 mmol) in EtOH (10mL) was added hydrazine (0.25 mL, 8.0 mmol). The resultant mixture wasstirred at room temperature for 3 hours. After removal of theprecipitate, the filtrate was concentrated. The residue was left in highvacuum overnight. Purification of the residue by flash chromatography(silica, 3:1-1:1 EtOAc:MeOH) afforded the amine linker (301 mg, 86%) asa white solid.

To a solution of the above activated mPEG(30K) (1.0 g, 0.033 mmol) inanhydrous CH₂Cl₂ (10 mL) were added diisopropylethylamine (58 μL, 0.33mmol), 4-dimethylaminopyridine (4 mg, 0.033 mmol) and the above aminelinker (64 mg, 0.31 mmol). The resultant mixture was stirred at roomtemperature for 15 hours. Ether (200 mL) was added to the reactionmixture. The precipitate was filtered, washed with ether and dried invacuo to afford a white powder (0.85 g, 85%).

To a solution of Boc protected mPEG(30K) (0.85 g, 0.028 mmol) inanhydrous CH₂Cl₂ (10 mL) at 0° C. was added trifluoroacetic acid (10mL). The resultant mixture was stirred at room temperature for 5 hours.Ether (200 mL) was added to the reaction mixture. The precipitate wasfiltered, washed with ether and dried in vacuo to afford a white powder(0.68 g, 80%).

Example 62

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 50A.

To a solution of mono-Boc phthalimide (2.1 g, 6.9 mmol) in pyridine (50mL) at 0° C. was added Boc₂O (3.3 g, 15.1 mmol). The resultant mixturewas heated to 60° C. overnight. After the solvent was removed in vacuo,the residue was diluted with EtOAc (200 mL) and washed with citric acid(5%, 200 mL), water (200 mL) and brine (200 mL), then dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 20:1-3:1 hexane:EtOAc)afforded product (2.37 g, 85%) as a yellow oil.

To a solution of di-Boc phthalimide (1.21 g, 2.98 mmol) in MeOH (15 mL)at 0° C. was added ammonia in MeOH (15 mL, 7 N, 105 mmol). The resultantmixture was stirred at room temperature overnight. The precipitate wasfiltered off and the filtrate was concentrated in vacuo. Purification ofthe residue by flash chromatography (silica, 10:1-6:4 EtOAc:MeOH)afforded the amine linker (0.61 g, 74%) as a white solid.

To a solution of mPEG(30K)-OH (1.0 g, 0.033 mmol) in anhydrous CH₂Cl₂(10 mL) was added p-nitrophenol chloro formate (60 mg, 0.28 mmol). Themixture was stirred at room temperature for 15 hours. Ether (200 mL) wasadded to precipitate mPEG(30K) product. The product was filtered, washedwith ether and dried in vacuo (1.0 g, 100%).

To a solution of the above activated mPEG(30K) (1.0 g, 0.033 mmol) inanhydrous CH₂Cl₂ (10 mL) were added diisopropylethylamine (58 μL, 0.33mmol), 4-dimethylaminopyridine (5 mg, 0.041 mmol) and the above diBocamine linker (90 mg, 0.33 mmol). The resultant mixture was stirred atroom temperature for 15 hours. Ether (200 mL) was added to the reactionmixture. The precipitate was filtered, washed with ether and dried invacuo to afford a white powder (0.82 g, 82%).

To a solution of Boc protected mPEG(30K) (0.82 g, 0.027 mmol) inanhydrous CH₂Cl₂ (8 mL) at 0° C. was added trifluoroacetic acid (8 mL).The resultant mixture was stirred at room temperature for 5 hours. Ether(200 mL) was added to the reaction mixture. The precipitate wasfiltered, washed with ether and dried in vacuo to afford a white powder(0.57 g, 70%).

Example 63

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 50B.

To a solution of mono-Boc phthalimide (1.5 g, 4.7 mmol) in pyridine at0° C. was added Boc₂O (2.2 g, 10.0 mmol). The resultant mixture washeated to 60° C. overnight. After the solvent was removed in vacuo, theresidue was diluted with EtOAc (200 mL) and washed with citric acid (5%,200 mL), water (200 mL) and brine (200 mL), then dried over anhydrousNa₂SO₄, filtered and concentrated in vacuo. Purification of the residueby flash chromatography (silica, 20:1-3:1 hexane:EtOAc) afforded product(1.6 g, 81%) as an oil.

To a solution of di-Boc phthalimide (1.5 g, 3.6 mmol) in MeOH (15 mL) at0° C. was added ammonia in MeOH (15 mL, 7 N, 105 mmol). The resultantmixture was stirred at room temperature overnight. The precipitate wasfiltered off and the filtrated was concentrated in vacuo. Purificationof the residue by flash chromatography (silica, 10:1-6:4 EtOAc:MeOH)afforded the amine linker (0.85 g, 82%) as a white solid.

To a solution of mPEG(30K)-OH (1.0 g, 0.033 mmol) in anhydrous CH₂Cl₂(10 mL) was added p-nitrophenol chloroformate (60 mg, 0.28 mmol). Themixture was stirred at room temperature for 15 hours. Ether (200 mL) wasadded. The precipitate was filtered, washed with ether and dried invacuo to afford product (1.0 g, 100%) as a white powder.

To a solution of the above activated mPEG(30K) (1.0 g, 0.033 mmol) inanhydrous CH₂Cl₂ (10 mL) were added diisopropylethylamine (58 μL, 0.33mmol), 4-dimethylaminopyridine (5 mg, 0.041 mmol) and the above di-Bocamine linker (100 mg, 0.34 mmol). The resultant mixture was stirred atroom temperature for 15 hours. Ether (200 mL) was added to the reactionmixture. The precipitate was filtered, washed with ether and dried invacuo to afford a white powder (0.89 g, 89%).

To a solution of Boc protected mPEG(30K) (0.89 g, 0.030 mmol) inanhydrous CH₂Cl₂ (8 mL) at 0° C. was added trifluoroacetic acid (8 mL).The resultant mixture was stirred at room temperature for 5 hours. Ether(200 mL) was added to the reaction mixture. The precipitate wasfiltered, washed with ether and dried in vacuo to afford a white powder(0.65 g, 73%).

Example 64

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 51A.

To a mixture of mPEG(30K) propionaldehyde (0.5 g, 0.0166 mmol) and aminelinker (73 mg, 0.25 mmol) in MeOH (10 mL) was added NaCNBH₃ (20 mg, 0.30mmol). The resultant mixture was stirred at room temperature for 48hours. After most solvent was removed, ether (200 mL) was added. Theprecipitate was filtered, washed with ether and dried in vacuo to afforda white powder (0.43 g, 86%).

To a solution of di-Boc protected mPEG(30K) (0.42 g, 0.014 mmol) inanhydrous CH₂Cl₂ (4 mL) at 0° C. was added trifluoroacetic acid (4 mL).The resultant mixture was stirred at room temperature for 8 hours. Ether(100 mL) was added to the reaction mixture. The precipitate wasfiltered, washed with ether and dried in vacuo to afford a white powder(0.35 g, 83%).

Example 65

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 51B.

To a stirred solution of mPEG(30K) (6.0 g, 0.2 mmol) and pyridine (0.1mL, 1.2 mmol) in CH₂Cl₂ (60 mL) at 0° C. is added Dess-Martinperiodinane (0.2 g, 0.47 mmol). The mixture is stirred at roomtemperature overnight. The reaction is then quenched with saturatedaqueous Na₂S₂O₃—NaHCO₃ (1:1, 100 mL) and extracted with CH₂Cl₂ (500mL×2). The organic layers are combined and washed with H₂O and brine,then dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo.The residue is dissolved in CH₂Cl₂ (50 mL). Ether (1 L) is added to thesolution. The precipitate is filtered, washed with ether and dried invacuo to afford a white powder (4.9 g, 82%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(73 mg, 0.25 mmol) in MeOH (10 mL) is added NaCNBH₃ (20 mg, 0.30 mmol).The resultant mixture is stirred at room temperature for 48 hours. Aftermost solvent is removed, ether (200 mL) is added. The precipitate isfiltered, washed with ether and dried in vacuo to afford a white powder(0.43 g, 85%).

To a solution of di-Boc protected mPEG(30K) (0.42 g, 0.014 mmol) inanhydrous CH₂Cl₂ (4 mL) at 0° C. is added trifluoroacetic acid (4 mL).The resultant mixture is stirred at room temperature for 8 hours. Ether(100 mL) is added to the reaction mixture. The precipitate is filtered,washed with ether and dried in vacuo to afford a white powder (0.35 g,83%).

Example 66

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 52A.

To a solution of mPEG(30K)-NH₂ (6.0 g, 0.2 mmol) in DMF (60 mL) areadded Boc-Ser-OH (205 mg, 1.0 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 190mg, 1.0 mmol) and N,N′-diisopropylethylamine (0.17 mL, 1.0 mmol). Themixture is stirred at room temperature for 10 h and diluted with EtOAc(500 mL). The resultant mixture is washed successively with saturatedaqueous NaHCO₃ (300 mL), H₂O (300 mL) and brine (300 mL), then driedover anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residueis dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. The precipitateis filtered, washed with ether and dried in vacuo to afford a whitepowder (5.1 g, 82%).

To a solution of the above mPEG(30K) (3.0 g, 0.1 mmol) in CH₂Cl₂ (15 mL)at 0° C. is added trifluoroacetic acid (15 mL). The resultant mixture isstirred at room temperature for 3 h and concentrated. To the residue isadded CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N in dioxane, 2mL). Ether (400 mL) is added to precipitate dihydroxylamine (2.6 g, 85%)as a white solid.

To a solution of the above mPEG(30K) (2.0 g, 0.067 mmol) in H₂O—CH₃CN(1:1, 20 mL) is added NaIO₄ (15 mg, 0.07 mmol). The mixture is stirredat room temperature for 4.0 h and diluted with CH₂Cl₂ (500 mL). Theresultant mixture is washed with H₂O (100 mL) and brine (100 mL), thendried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Theresidue is dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. Theprecipitate is filtered, washed with ether and dried in vacuo to afforda white powder (1.8 g, 90%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(73 mg, 0.25 mmol) in MeOH (10 mL) is added NaCNBH₃ (20 mg, 0.30 mmol).The resultant mixture is stirred at room temperature for 48 h. Aftermost solvent is removed, ether (200 mL) was added. The precipitate isfiltered, washed with ether and dried in vacuo to afford a white powder(0.43 g, 86%).

To a solution of diBoc protected mPEG(30K) (0.42 g, 0.014 mmol) inanhydrous CH₂Cl₂ (4 mL) at 0° C. is added trifluoroacetic acid (4 mL).The resultant mixture was stirred at room temperature for 8 h. Ether(100 mL) is added to the reaction mixture. The precipitate is filtered,washed with ether and dried in vacuo to afford a white powder (0.35 g,83%).

Example 67

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 52B.

To a mixture of mPEG propionaldehyde (30 K, 0.5 g, 0.0166 mmol) andamine linker (70 mg, 0.25 mmol) in MeOH (10 mL) was added NaCNBH₃ (20mg, 0.30 mmol). The resultant mixture was stirred at room temperaturefor 48 hours. After most solvent was removed, ether (200 mL) was added.The precipitate was filtered, washed with ether and dried in vacuo toafford a white powder (0.40 g, 80%).

To a solution of di-Boc protected mPEG(30K) (0.40 g, 0.013 mmol) inanhydrous CH₂Cl₂ (4 mL) at 0° C. was added trifluoroacetic acid (4 mL).The resultant mixture was stirred at room temperature for 8 hours. Ether(100 mL) was added to the reaction mixture. The precipitate wasfiltered, washed with ether and dried in vacuo to afford a white powder(0.35 g, 87%).

Example 68

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 53A.

To a stirred solution of mPEG(30K) (6.0 g, 0.2 mmol) and pyridine (0.1mL, 1.2 mmol) in CH₂Cl₂ (60 mL) at 0° C. is added Dess-Martinperiodinane (0.2 g, 0.47 mmol). The mixture is stirred at roomtemperature overnight. The reaction is then quenched with saturatedaqueous Na₂S₂O₃—NaHCO₃ (1:1, 100 mL) and extracted with CH₂Cl₂ (500mL×2). The organic layers are combined and washed with H₂O and brine,then dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo.The residue is dissolved in CH₂Cl₂ (50 mL). Ether (1 L) was added to thesolution. The precipitate was filtered, washed with ether and dried invacuo to afford a white powder (4.9 g, 82%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(70 mg, 0.25 mmol) in MeOH (10 mL) is added NaCNBH₃ (20 mg, 0.30 mmol).The resultant mixture is stirred at room temperature for 48 hours. Aftermost solvent is removed, ether (200 mL) is added. The precipitate isfiltered, washed with ether and dried in vacuo to afford a white powder(0.40 g, 80%).

To a solution of di-Boc protected mPEG(30K) (0.40 g, 0.013 mmol) inanhydrous CH₂Cl₂ (4 mL) at 0° C. is added trifluoroacetic acid (4 mL).The resultant mixture is stirred at room temperature for 8 hours. Ether(100 mL) is added to the reaction mixture. The precipitate is filtered,washed with ether and dried in vacuo to afford a white powder (0.35 g,87%).

Example 69

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 53B.

To a solution of mPEG(30K)-NH₂ (6.0 g, 0.2 mmol) in DMF (60 mL) areadded Boc-Ser-OH (205 mg, 1.0 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 190mg, 1.0 mmol) and N,N′-diisopropylethylamine (0.17 mL, 1.0 mmol). Themixture is stirred at room temperature for 10 h and diluted with EtOAc(500 mL). The resultant mixture is washed successively with saturatedaqueous NaHCO₃ (300 mL), H₂O (300 mL) and brine (300 mL), then driedover anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residueis dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. The precipitateis filtered, washed with ether and dried in vacuo to afford a whitepowder (5-1 g, 82%).

To a solution of the above mPEG(30K) (3.0 g, 0.1 mmol) in CH₂Cl₂ (15 mL)at 0° C. is added trifluoroacetic acid (15 mL). The resultant mixture isstirred at room temperature for 3 h and concentrated. To the residue isadded CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N in dioxane, 2mL). Ether (400 mL) is added to precipitate dihydroxylamine (2.6 g, 85%)as a white solid.

To a solution of the above mPEG(30K) (2.0 g, 0.067 mmol) in H₂O—CH₃CN(1:1, 20 mL) is added NaIO₄ (15 mg, 0.07 mmol). The mixture is stirredat room temperature for 4.0 h and diluted with CH₂Cl₂ (500 mL). Theresultant mixture is washed with H₂O (100 mL) and brine (100 mL), thendried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Theresidue is dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. Theprecipitate is filtered, washed with ether and dried in vacuo to afforda white powder (1.8 g, 90%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(70 mg, 0.25 mmol) in MeOH (10 mL) is added NaCNBH₃ (20 mg, 0.30 mmol).The resultant mixture is stirred at room temperature for 48 h. Aftermost solvent is removed, ether (200 mL) is added. The precipitate isfiltered, washed with ether and dried in vacuo to afford a white powder(0.40 g, 80%).

To a solution of di-Boc protected mPEG(30K) (0.40 g, 0.013 mmol) inanhydrous CH₂Cl₂ (4 mL) at 0° C. is added trifluoroacetic acid (4 mL).The resultant mixture is stirred at room temperature for 8 h. Ether (100mL) is added to the reaction mixture. The precipitate is filtered,washed with ether and dried in vacuo to afford a white powder (0.35 g,87%).

Example 70

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 54A.

To a mixture of mPEG(30K) propionaldehyde (0.5 g, 0.0166 mmol) and aminelinker (40 mg, 0.16 mmol) in MeOH (10 mL) was added NaCNBH₃ (12 mg, 0.17mmol). The resultant mixture was stirred at room temperature for 60hours. After most solvent was removed, the residue was dissolved inCH₂Cl₂ (200 mL) and washed with citric acid (5%, 100 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated invacuo to afford a white solid (0.42 g, 84%).

To a mixture of mPEG(30K) phthalimide (0.4 g, 0.013 mmol) in MeOH (4 mL)was added H₂NNH₂ (4.2 SL, 0.13 mmol). The mixture was stirred at 45° C.for 1.0 hour and concentrated. The residue was dissolved in CH₂Cl₂ (100mL) and washed with HCl (0.1 N, 100 mL). The aqueous layer was extractedwith CH₂Cl₂ (100 mL). The organic layers were combined and washed withH₂O, then dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was dissolved in CH₂Cl₂ (5 mL). Ether (200 mL) was added toprecipitate the hydroxylamine product (0.34 g, 85%) as a white powder.

Example 71

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 54B.

To a stirred solution of mPEG(30K) (6.0 g, 0.2 mmol) and pyridine (0.1mL, 1.2 mmol) in CH₂Cl₂ (60 mL) at 0° C. is added Dess-Martinperiodinane (0.2 g, 0.47 mmol). The mixture is stirred at roomtemperature overnight. The reaction is then quenched with saturatedaqueous Na₂S₂O₃—NaHCO₃ (1:1, 100 mL) and extracted with CH₂Cl₂ (500mL×2). The organic layers are combined and washed with H₂O and brine,then dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo.The residue is dissolved in CH₂Cl₂ (50 mL). Ether (1 L) is added to thesolution. The precipitate is filtered, washed with ether and dried invacuo to afford a white powder (4.9 g, 82%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(40 mg, 0.16 mmol) in MeOH (10 mL) is added NaCNBH₃ (12 mg, 0.17 mmol).The resultant mixture is stirred at room temperature for 60 hours. Aftermost solvent is removed, the residue is dissolved in CH₂Cl₂ (200 mL) andwashed with citric acid (5%, 100 mL). The organic layer is dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to afford a whitesolid (0.42 g, 84%).

To a mixture of mPEG(30K) phthalimide (0.4 g, 0.013 mmol) in MeOH (4 mL)is added H₂NNH₂ (4.2 μL, 0.13 mmol). The mixture is stirred at 45° C.for 1.0 hour and concentrated. The residue is dissolved in CH₂Cl₂ (100mL) and washed with HCl (0.1 N, 100 mL). The aqueous layer is extractedwith CH₂Cl₂ (100 mL). The organic layers are combined and washed withH₂O, then dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue is dissolved in CH₂Cl₂ (5 mL). Ether (200 mL) is added toprecipitate the hydroxylamine product (0.34 g, 85%) as a white powder.

Example 72

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 55.

To a solution of mPEG(30K)-NH₂ (6.0 g, 0.2 mmol) in DMF (60 mL) areadded Boc-Ser-OH (205 mg, 1.0 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 190mg, 1.0 mmol) and N,N′-diisopropylethylamine (0.17 mL, 1.0 mmol). Themixture is stirred at room temperature for 10 h and diluted with EtOAc(500 mL). The resultant mixture is washed successively with saturatedaqueous NaHCO₃ (300 mL), H₂O (300 mL) and brine (300 mL), then driedover anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residueis dissolved in CH₂CH₂ (50 mL). Ether (700 mL) is added. The precipitateis filtered, washed with ether and dried in vacuo to afford a whitepowder (5-1 g, 82%).

To a solution of the above mPEG(30K) (3.0 g, 0.1 mmol) in CH₂Cl₂ (15 mL)at 0° C. is added trifluoroacetic acid (15 mL). The resultant mixture isstirred at room temperature for 3 h and concentrated. To the residue isadded CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N in dioxane, 2mL). Ether (400 mL) is added to precipitate dihydroxylamine (2.6 g, 85%)as a white solid.

To a solution of the above mPEG(30K) (2.0 g, 0.067 mmol) in H₂O—CH₃CN(1:1, 20 mL) is added NaIO₄ (15 mg, 0.07 mmol). The mixture is stirredat room temperature for 4.0 h and diluted with CH₂Cl₂ (500 mL). Theresultant mixture is washed with H₂O (100 mL) and brine (100 mL), thendried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Theresidue is dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. Theprecipitate is filtered, washed with ether and dried in vacuo to afforda white powder (1.8 g, 90%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(40 mg, 0.16 mmol) in MeOH (10 mL) is added NaCNBH₃ (12 mg, 0.17 mmol).The resultant mixture is stirred at room temperature for 60 h. Aftermost solvent is removed, the residue is dissolved in CH₂Cl₂ (200 mL) andwashed with citric acid (5%, 100 mL). The organic layer is dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to afford a whitesolid (0.42 g, 84%).

To a mixture of mPEG(30K) phthalimide (0.4 g, 0.013 mmol) in MeOH (4 mL)is added H₂NNH₂ (4.2 μL, 0.13 mmol). The mixture is stirred at 45° C.for 1.0 h and concentrated. The residue is dissolved in CH₂Cl₂ (100 mL)and washed with HCl (0.1 N, 100 mL). The aqueous layer is extracted withCH₂Cl₂ (100 mL). The organic layers are combined and washed with H₂O,then dried over anhydrous Na₂SO₄, filtered and concentrated. The residueis dissolved in CH₂Cl₂ (5 mL). Ether (200 mL) is added to precipitatethe hydroxylamine product (0.34 g, 85%) as a white powder.

Example 73

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 56A.

To a mixture of mPEG(30K) propionaldehyde (0.5 g, 0.0166 mmol) and aminelinker (40 mg, 0.16 mmol) in MeOH (10 mL) is added NaCNBH₃ (12 mg, 0.17mmol). The resultant mixture is stirred at room temperature for 60hours. After most solvent is removed, the residue is dissolved in CH₂Cl₂(200 mL) and washed with citric acid (5%, 100 mL). The organic layer isdried over anhydrous Na₂SO₄, filtered and concentrated in vacuo toafford a white solid (0.41 g, 82%).

To a mixture of mPEG(30K) phthalimide (0.4 g, 0.013 mmol) in MeOH (4 mL)is added H₂NNH₂ (4.2 μL, 0.13 mmol). The mixture is stirred at 45° C.for 1.0 hour and concentrated. The residue is dissolved in CH₂Cl₂ (100mL) and washed with HCl (0.1 N, 100 mL). The aqueous layer is extractedwith CH₂Cl₂ (100 mL). The organic layers are combined and washed withH₂O, then dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was dissolved in CH₂Cl₂ (5 mL). Ether (200 mL) is added toprecipitate the hydroxylamine product (0.34 g, 83%) as a white powder.

Example 74

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 56B.

To a stirred solution of mPEG(30K) (6.0 g, 0.2 mmol) and pyridine (0.1mL, 1.2 mmol) in CH₂Cl₂ (60 mL) at 0° C. is added Dess-Martinperiodinane (0.2 g, 0.47 mmol). The mixture is stirred at roomtemperature overnight. The reaction is then quenched with saturatedaqueous Na₂S₂O₃—NaHCO₃ (1:1, 100 mL) and extracted with CH₂Cl₂ (500mL×2). The organic layers are combined and washed with H₂O and brine,then dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo.The residue is dissolved in CH₂Cl₂ (50 mL). Ether (1 L) is added to thesolution. The precipitate is filtered, washed with ether and dried invacuo to afford a white powder (4.9 g, 82%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(40 mg, 0.16 mmol) in MeOH (10 mL) is added NaCNBH₃ (12 mg, 0.17 mmol).The resultant mixture is stirred at room temperature for 60 hours. Aftermost solvent is removed, the residue is dissolved in CH₂Cl₂ (200 mL) andwashed with citric acid (5%, 100 mL). The organic layer is dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to afford a whitesolid (0.41 g, 82%).

To a mixture of mPEG(30K) phthalimide (0.4 g, 0.013 mmol) in MeOH (4 mL)is added H₂NNH₂ (4.2 μL, 0.13 mmol). The mixture is stirred at 45° C.for 1.0 hour and concentrated. The residue is dissolved in CH₂Cl₂ (100mL) and washed with HCl (0.1 N, 100 mL). The aqueous layer is extractedwith CH₂Cl₂ (100 mL). The organic layers are combined and washed withH₂O, then dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue is dissolved in CH₂Cl₂ (5 mL). Ether (200 mL) is added toprecipitate the hydroxylamine product (0.34 g, 83%) as a white powder.

Example 75

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 57.

To a solution of mPEG(30K)-NH₂ (6.0 g, 0.2 mmol) in DMF (60 mL) areadded Boc-Ser-OH (205 mg, 1.0 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 190mg, 1.0 mmol) and N,N′-diisopropylethylamine (0.17 mL, 1.0 mmol). Themixture is stirred at room temperature for 10 h and diluted with EtOAc(500 mL). The resultant mixture is washed successively with saturatedaqueous NaHCO₃ (300 mL), H₂O (300 mL) and brine (300 mL), then driedover anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residueis dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. The precipitateis filtered, washed with ether and dried in vacuo to afford a whitepowder (5.1 g, 82%).

To a solution of the above mPEG(30K) (3.0 g, 0.1 mmol) in CH₂Cl₂ (15 mL)at 0° C. is added trifluoroacetic acid (15 mL). The resultant mixture isstirred at room temperature for 3 h and concentrated. To the residue isadded CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N in dioxane, 2mL). Ether (400 mL) is added to precipitate dihydroxylamine (2.6 g, 85%)as a white solid.

To a solution of the above mPEG(30K) (2.0 g, 0.067 mmol) in H₂O—CH₃CN(1:1, 20 mL) is added NaIO₄ (15 mg, 0.07 mmol). The mixture is stirredat room temperature for 4.0 h and diluted with CH₂Cl₂ (500 mL). Theresultant mixture is washed with H₂O (100 mL) and brine (100 mL), thendried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Theresidue is dissolved in CH₂Cl₂ (50 mL). Ether (700 mL) is added. Theprecipitate is filtered, washed with ether and dried in vacuo to afforda white powder (1.8 g, 90%).

To a mixture of mPEG(30K) aldehyde (0.5 g, 0.0166 mmol) and amine linker(40 mg, 0.16 mmol) in MeOH (10 mL) is added NaCNBH₃ (12 mg, 0.17 mmol).The resultant mixture is stirred at room temperature for 60 h. Aftermost solvent is removed, the residue is dissolved in CH₂Cl₂ (200 mL) andwashed with citric acid (5%, 100 mL). The organic layer is dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to afford a whitesolid (0.41 g, 82%).

To a mixture of mPEG(30K) phthalimide (0.4 g, 0.013 mmol) in MeOH (4 mL)is added H₂NNH₂ (4.2 μL, 0.13 mmol). The mixture is stirred at 45° C.for 1.0 h and concentrated. The residue is dissolved in CH₂Cl₂ (100 mL)and washed with HCl (0.1 N, 100 mL). The aqueous layer is extracted withCH₂Cl₂ (100 mL). The organic layers are combined and washed with H₂O,then dried over anhydrous Na₂SO₄, filtered and concentrated. The residueis dissolved in CH₂Cl₂ (5 mL). Ether (200 mL) is added to precipitatethe hydroxylamine product (0.34 g, 83%) as a white powder.

Example 76

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 58A.

Synthesis of mPEG(5K)-OMs

To a solution of mPEG(5K)-OH (1.0 g, 0.2 mmol) in CH₂Cl₂ (40 mL) wereadded triethylamine (110 μL, 0.79 mmol) and MsCl (50 μL, 0.64 mmol). Themixture was stirred at room temperature for 10 hours and concentrated.The crude product (1.0 g) was directly used for the next step withoutpurification.

Synthesis of mPEG(5K)-O—NHBoc

To a solution of the crude mPEG(5K)-OMs (1.0 g, 0.2 mmol) in CH₂Cl₂ (10mL) were added t-butyl-N-hydroxycarbamate (0.3 g, 2.2 mmol) andtriethylamine (0.4 mL, 2.9 mmol). The resultant mixture was stirred at45° C. for 10 hours and cooled to the room temperature. Ether (200 mL)was added. The precipitate was filtered, washed and dried in vacuo toafford product (0.42, 42%) as a white solid.

Synthesis of mPEG(5K)-O—NH₃ ⁺Cl⁻

To a solution of mPEG(5K)-ONHBoc (0.2 g, 0.04 mmol) in CH₂Cl₂ (3 mL) at° C. was added trifluoroacetic acid (7 mL). The resultant mixture wasstirred at room temperature for 1 hour and concentrated. To the residueis added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N in dioxane,2 mL). Ether (300 mL) is added to precipitate PEG dihydroxylaminederivative (170 mg, 85%) as a white solid.

Example 77

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 58B.

Synthesis of mPEG(30K)-OTf

To a solution of mPEG(30K)-OH (3.0 g, 0.1 mmol) in CH₂Cl₂ (30 mL) areadded 2,6-lutidine (60 μL, 0.5 mmol) and Tf₂O (65 μL, 0.4 mmol). Themixture is stirred at room temperature for 10 hours. Ether (400 mL) isadded to the mixture. The precipitate is filtered, washed with ether anddried in vacuo to afford a white powder (2.7 g, 90%).

Synthesis of mPEG(30K)-O—NHBoc

To a solution of mPEG(30K)-OTf (2.5 g, 0.083 mmol) in CH₂Cl₂ (25 mL) areadded t-butyl N-hydroxycarbamate (110 mg, 0.84 mmol) anddiisopropylethylamine (0.2 mL, 1.1 mmol). The mixture is stirred at roomtemperature overnight. Ether (200 mL) is added to precipitate theproduct (2.2 g, 88%) as a white powder.

Synthesis of mPEG(30K)-O—NH₃ ⁺Cl⁻

To a solution of the above mPEG(30K)-ONHBoc (2.0 g, 0.067 mmol) inCH₂Cl₂ (15 mL) at 0° C. is added trifluoroacetic acid (15 mL). Theresultant mixture is stirred at room temperature for 3 hours andconcentrated. To the residue is added CH₂Cl₂ (5 mL) followed by theaddition of HCl (4 N in dioxane, 2 mL). Ether (300 mL) is added toprecipitate PEG dihydroxylamine derivative (1.72 g, 86%) as a whitesolid.

Example 78

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 59A.

To a solution of 2-(2-hydroxyethoxy)phthalimide (0.5 g, 2.4 mmol) inCH₂Cl₂ (20 mL) was added phosgene (20% in toluene, 8.0 mL, 15.0 mmol).The reaction mixture was stirred at room temperature for 10 hour andconcentrated in vacuo. The residue (0.45 g, 70%) was used directly forthe next reaction without purification.

To a mixture of mPEG(30K)-NH₂ (3 g, 01 mmol) and chloroformate linker(0.27 g, 1.0 mmol) in CH₂Cl₂ (30 mL) was added diisopropylethylamine(0.2 mL, 1.1 mmol). The resultant mixture was stirred at roomtemperature for 15 hours. Ether (500 mL) was added to the mixture. Theprecipitate was filtered, washed and dried in vacuo to afford product(2.7 g, 90%) as a white solid.

To a solution of mPEG(30K) phthalimide (2.1 g, 0.07 mmol) in MeOH (15mL) was added ammonia (7 N in methanol, 15 mL). The resultant mixturewas stirred at room temperature for 15 hours and concentrated. To theresidue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N indioxane, 1 mL). Ether (300 mL) was added to precipitate the product (2.4g, 89%) as a white solid.

Example 79

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 59B.

To a solution of (Boc-aminooxy)acetic acid (3.0 g, 16 mmol) in CH₂Cl₂(80 mL) was added N—N′-diisopropylcarbodiimide (DIC, 1.3 mL, 8 mmol).The mixture was stirred at room temperature for 1 hour and concentratedin vacuo. The crude product (4.9 g, 84%) was directly used for the nextstep without further purification.

To a solution of anhydride (7.3 g, 20 mmol) in DMF (20 mL) was addedmPEG(5K)-NH₂ (20 g, 4 mmol). The mixture was stirred at room temperaturefor 10 hours and diluted with H₂O (200 mL). The mixture was extractedwith CH₂Cl₂ (500 mL). The organic layer was washed with H₂O and brine(100 mL), then dried over anhydrous Na₂SO₄, filtered and concentrated invacuo. To the residue was added CH₂Cl₂ (10 mL) followed by ether (500mL). The precipitate was filtered, washed with ether and dried in vacuoto afford product (19.8 g. 99%) as a white powder.

To a solution of Boc protected mPEG(5K) (1.0 g, 0.2 mmol) in CH₂Cl₂ (5mL) was added trifluoroacetic acid (5 mL). The resultant mixture wasstirred at room temperature for 1 hour and concentrated. To the residuewas added CH₂Cl₂ (2 mL) followed by the addition of HCl (4 N in dioxane,0.1 mL) and ether (40 mL). The precipitate was filtered, washed withether and dried in vacuo to afford product (0.75 g, 75%).

Example 80

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 60A.

To a solution of mPEG(30K)-OH (4.5 g, 0.15 mmol) in CH₂Cl₂ (50 mL) wasadded phosgene (20% in toluene, 1.6 mL, 3.0 mmol). The reaction mixturewas stirred at room temperature for 10 hours and concentrated in vacuo.The residue (4.2 g, 93%) was used directly for the next reaction withoutpurification.

To a mixture of the above activated mPEG(30 K) II (4.2 g, 0.14 mmol) andthe amine linker VIII (67 mg, 0.28 mmol) in DMF-CH₂Cl₂ (10 mL, 1:2) wereadded diisopropylethylamine (75 μL, 0.42 mmol). After the resultantmixture was stirred at room temperature for 15 hours, Ether (200 mL) wasadded. The precipitate was filtered, washed with ether and dried invacuo to afford product (3.8 g, 90%) as a white powder.

To a solution of mPEG(30K) phthalimide III (3.5 g, 0.12 mmol) in MeOH(15 mL) was added ammonia (7 N in methanol, 15 mL). The resultantmixture was stirred at room temperature for 15 hours and concentrated.To the residue was added CH₂Cl₂ (5 mL) followed by the addition of HCl(4 N in dioxane, 1 mL). Ether (300 mL) was added to precipitate theproduct (3.0 g, 86%) as a white solid.

To a solution of t-butyl 2-hydroxyethylcarbamate (2.8 mL, 18 mmol) inTHF (60 mL) were added N-hydroxyphthalimide (5.8 g, 36 mmol),triphenylphosphine (3.6 g, 27 mmol). The reaction mixture was stirred atroom temperature for 10 minutes and then cooled to 0° C.Diisopropylazodicarboxylate (DIAD, 3.6 mL, 19 mmol) was added dropwisevia syringe over 1 hour. The icebath was removed and the mixture wasstirred overnight and concentrated. The white solid dissolved in ethylacetate (100 mL). The reaction mixture was washed successively withsaturated aqueous sodium bicarbonate solution (2×50 mL), deionized water(50 mL) and brine (50 mL), then dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. The crude product was purified by flashchromatography using a Biotage Inc. HORIZON™ chromatography system toafford the title compound with impurities (12 g, 206%) as a white solid.

To a solution of the crude Boc-protected linker (12 g) in CH₂Cl₂ (5 mL)was added trifluoroacetic acid (5 mL). The resultant mixture was stirredat room temperature for 1 hour and concentrated. To the residue wasadded HCl (4 N in dioxane, 1.5 mL) followed by the addition of Et₂O (150mL). The precipitate was filtered, washed with ether and dried in vacuoto afford the amine linker (3.0 g, 68% for two steps) as a white solid.

Example 81

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 60B.

To a solution of mPEG(5K)-OH (10 g, 2.0 mmol), Ph₃P (790 mg, 3.0 mmol)and N-hydroxyphthalimide (0.49 g, 3.0 mmol) in CH₂Cl₂-THF (2:3, 45 mL)at 0° C. was added diisopropyl azodicarboxylate (DIAD, 409 μL, 2.0mmol). After the mixture was stirred at room temperature for 15 hours,ether (1 L) was added to the mixture. The precipitate was filtered,washed with ether and dried in vacuo to afford mPEG(5K)-phthalimide (9.8g, 98%) as a white powder.

Synthesis of mPEG(5K)-O—NH₂

To a solution of mPEG(5K) phthalimide (3 g, 0.6 mmol) in MeOH (25 mL)was added ammonia (7 N in methanol, 25 mL). The resultant mixture wasstirred at room temperature for 15 hours and concentrated. To theresidue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N indioxane, 1 mL). Ether (300 mL) was added to precipitate hydroxylamine(2.5 g, 83%) as a white solid.

To a solution of mPEG(30K)-OH (6 g, 0.2 mmol), Ph₃P (80 mg, 0.3 mmol)and N-hydroxyphthalimide (49 mg, 0.3 mmol) in CH₂Cl₂-THF (2:3, 45 mL) at0° C. was added diisopropyl azodicarboxylate (DIAD, 41 μL, 0.2 mmol).The mixture was stirred at room temperature for 15 hours. Ether (200 mL)was added to the mixture. The precipitate was washed with ether anddried in vacuo to afford mPEG(30K)-phthalimide product (5.8 g, 96%) as awhite powder.

Synthesis of mPEG(30K)-O—NH₂

To a solution of mPEG(30K) phthalimide (3 g, 0.1 mmol) in MeOH (20 mL)was added ammonia (7 N in methanol, 20 mL). The resultant mixture wasstirred at room temperature for 15 hours and concentrated. To theresidue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N indioxane, 1 mL). Ether (300 mL) was added to precipitate mPEG(30K)-ONH₂(2.6 g, 87%) as a white solid.

Example 82

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 61A.

To a solution of 2-(2-(2-aminoethoxy)ethoxy)ethanamine (5.0 g, 33.8mmol) in DMF (100 mL) were added (Boc-aminooxy)acetic acid (14.2 g, 74.2mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC,28.5 g, 0.15 mol) and N,N′-diisopropylethylamine (26 mL, 0.15 mol). Themixture was stirred at room temperature for 10 hours and diluted withEtOAc (500 mL). The resultant mixture was washed successively withsaturated aqueous NaHCO₃ (300 mL), H₂O (300 mL) and brine (300 mL), thendried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Thecrude product (14.2 g, 85%) was used directly for the next reactionwithout purification.

To a solution of the above diBoc linker (3.0 g, 6.1 mmol) in CH₂Cl₂ (15mL) at 0° C. was added trifluoroacetic acid (15 mL). The resultantmixture was stirred at room temperature for 3 hours and concentrated. Tothe residue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 Nin dioxane, 2 mL). Ether (400 mL) was added to precipitatedihydroxylamine (1.47 g, 82%) as a white solid.

Example 83

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 61B.

To a solution of tri(ethylene glycol) (1.5 g, 10 mmol) in THF (100 mL)at 0° C. were added Ph₃P (8.0 g, 30 mmol) and N-hydroxylphthalimide (4.9g, 30 mmol). To the mixture was slowly added diisopropylazodicarboxylate (DIAD, 4.08 mL, 20 mmol). The resultant mixture wasstirred at 0° C. for 4 hours and room temperature for 2 days. Ether (25mL) was added to the reaction mixture. The precipitate was washed withether and dried in vacuo to afford di-phthalimide product (3.72 g, 82%)as a white powder.

To a solution of diphthalimide (2.2 g, 5.0 mmol) in MeOH (20 mL) wasadded ammonia (7 N in methanol, 20 mL). The resultant mixture wasstirred at room temperature for 15 hours and concentrated. To theresidue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N indioxane, 1 mL). Ether (300 mL) was added to precipitate tri(ethyleneglycol) dihydroxylamine linker (1.1 g, 87%) as a white solid.

Example 84

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 61C.

To a solution of tetra(ethylene glycol (1.94 g, 10 mmol) in THF (100 mL)at 0° C. were added Ph₃P (8.0 g, 30 mmol) and N-hydroxylphthalimide (4.9g, 30 mmol). To the mixture was slowly added diisopropylazodicarboxylate (DIAD, 4.08 mL, 20 mmol). The resultant mixture wasstirred at 0° C. for 4 hours and room temperature for 2 days. Ether (25mL) was added to the reaction mixture. The precipitate was washed withether and dried in vacuo to afford di-phthalimide product (3.58 g, 74%)as a white powder.

To a solution of diphthalimide (2.42 g, 5.0 mmol) in MeOH (20 mL) wasadded ammonia (7 N in methanol, 20 mL). The resultant mixture wasstirred at room temperature for 15 hours and concentrated. To theresidue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N indioxane, 1 mL). Ether (300 mL) was added to precipitate tetra(ethyleneglycol) dihydroxylamine linker (1.27 g, 85%) as a white solid.

Example 85

This example details the synthesis of the mPEG-hydroxylamine presentedin FIG. 62A.

To a solution of hexa(ethylene glycol (2.82 g, 10 mmol) in THF (100 mL)at 0° C. were added Ph₃P (8.0 g, 30 mmol) and N-hydroxylphthalimide (4.9g, 30 mmol). To the mixture was slowly added diisopropylazodicarboxylate (DIAD, 4.08 mL, 20 mmol). The resultant mixture wasstirred at 0° C. for 4 hours and room temperature for 2 days. Ether (25mL) was added to the reaction mixture. The precipitate was washed withether and dried in vacuo to afford di-phthalimide product (4.40 g, 77%)as a white powder.

To a solution of diphthalimide (2.86 g, 5.0 mmol) in MeOH (20 mL) wasadded ammonia (7 N in methanol, 20 mL). The resultant mixture wasstirred at room temperature for 15 hours and concentrated. To theresidue was added CH₂Cl₂ (5 mL) followed by the addition of HCl (4 N indioxane, 1 mL). Ether (300 mL) was added to precipitate hexa(ethyleneglycol) dihydroxylamine linker (1.68 g, 87%) as a white solid.

Example 86

This example details the synthesis of the mPEG compound presented inFIG. 62B.

To a solution of mPEG-OH (30 K, 3.0 g, 0.1 mmol) in anhydrous CH₂Cl₂ (30mL) was added diphosgene (63 μL, 0.5 mmol). The mixture was stirred atroom temperature overnight. Ether (700 mL) was added to precipitatemPEG. The product was filtered, washed with ether and dried in vacuo(3.0 g, 100%).

The following examples describe methods to measure and compare the invitro and in vivo activity of a modified therapeutically activenon-natural amino acid polypeptide to the in vitro and in vivo activityof a therapeutically active natural amino acid polypeptide.

Example 87 Cell Binding Assays

Cells (3×10⁶) are incubated in duplicate in PBS/1% BSA (100 μl) in theabsence or presence of various concentrations (volume: 10 μl) ofunlabeled GH, hGH or GM-CSF and in the presence of ¹²⁵I-GH (approx.100,000 cpm or 1 ng) at 0° C. for 90 minutes (total volume: 120 μl).Cells are then resuspended and layered over 200 μl ice cold FCS in a 350μl plastic centrifuge tube and centrifuged (1000 g; 1 minute). Thepellet is collected by cutting off the end of the tube and pellet andsupernatant counted separately in a gamma counter (Packard).

Specific binding (cpm) is determined as total binding in the absence ofa competitor (mean of duplicates) minus binding (cpm) in the presence of100-fold excess of unlabeled GH (non-specific binding). The non-specificbinding is measured for each of the cell types used. Experiments are runon separate days using the same preparation of ¹²⁵I-GH and shoulddisplay internal consistency. ¹²⁵I-GH demonstrates binding to the GHreceptor-producing cells. The binding is inhibited in a dose dependentmanner by unlabeled natural GH or hGH, but not by GM-CSF or othernegative control. The ability of hGH to compete for the binding ofnatural ¹²⁵I-GH, similar to natural GH, suggests that the receptorsrecognize both forms equally well.

Example 88 In Vivo Studies of PEGylated hGH

PEG-hGH, unmodified hGH and buffer solution are administered to mice orrats. The results will show superior activity and prolonged half life ofthe PEGylated hGH of the present invention compared to unmodified hGHwhich is indicated by significantly increased bodyweight.

Example 89 Measurement of the In Vivo Half-Life of Conjugated andNon-Conjugated hGH and Variants Thereof

All animal experimentation is conducted in an AAALAC accredited facilityand under protocols approved by the Institutional Animal Care and UseCommittee of St. Louis University. Rats are housed individually in cagesin rooms with a 12-hour light/dark cycle. Animals are provided access tocertified Purina rodent chow 5001 and water ad libitum. Forhypophysectomized rats, the drinking water additionally contains 5%glucose.

Example 90 Pharmacokinetic Studies

The quality of each PEGylated mutant hGH was evaluated by three assaysbefore entering animal experiments. The purity of the PEG-hGH wasexamined by running a 4-12% acrylamide NuPAGE Bis-Tris gel with MES SDSrunning buffer under non-reducing conditions (Invitrogen, Carlsbad,Calif.). The gels were stained with Coomassie blue. The PEG-hGH band wasgreater than 95% pure based on densitometry scan. The endotoxin level ineach PEG-hGH was tested by a kinetic LAL assay using the KTA² kit fromCharles River Laboratories (Wilmington, Mass.), and was less than 5 EUper dose. The biological activity of the PEG-hGH was assessed with aIM-9 pSTAT5 bioassay, and the EC₅₀ value confirmed to be less than 15nM.

Pharmacokinetic properties of PEG-modified growth hormone compounds werecompared to each other and to nonPEGylated growth hormone in maleSprague-Dawley rats (261-425 g) obtained from Charles RiverLaboratories. Catheters were surgically installed into the carotidartery for blood collection. Following successful catheter installation,animals were assigned to treatment groups (three to six per group) priorto dosing. Animals were dosed subcutaneously with 1 mg/kg of compound ina dose volume of 0.41-0.55 ml/kg. Blood samples were collected atvarious time points via the indwelling catheter and into EDTA-coatedmicrofuge tubes. Plasma was collected after centrifugation, and storedat −80° C. until analysis. Compound concentrations were measured usingantibody sandwich growth hormone ELISA kits from either BioSourceInternational (Camarillo, Calif.) or Diagnostic Systems Laboratories(Webster, Tex.). Concentrations were calculated using standardscorresponding to the analog that was dosed. Pharmacokinetic parameterswere estimated using the modeling program WinNonlin (Pharsight, version4.1). Noncompartmental analysis with linear-up/log-down trapezoidalintegration was used, and concentration data was uniformly weighted.

Plasma concentrations were obtained at regular intervals following asingle subcutaneous dose in rats. Rats (n=3-6 per group) were given asingle bolus dose of 1 mg/kg protein. hGH wild-type protein (WHO hGH),His-tagged hGH polypeptide (his-hGH), or His-tagged hGH polypeptidecomprising non-natural amino acid p-acetyl-phenylalanine covalentlylinked to 30 kDa PEG at each of six different positions were compared toWHO hGH and (his)-hGH. Plasma samples were taken over the regular timeintervals and assayed for injected compound as described. The tablebelow shows the pharmacokinetic parameter values for single-doseadministration of the various hGH polypeptides. Concentration vs timecurves were evaluated by noncompartmental analysis (Pharsight, version4.1). Values shown are averages (+/−standard deviation). Cmax: maximumconcentration; terminal_(t1/2): terminal half-life; AUC_(0->inf): areaunder the concentration-time curve extrapolated to infinity; MRT: meanresidence time; Cl/f: apparent total, plasma clearance; Vz/f: apparentvolume of distribution during terminal phase. 30 KPEG-pAF92 (his)_(h)GHwas observed to dramatically extended circulation, increase serumhalf-life, and increase bioavailability compared to control hGH

TABLE Pharmacokinetic parameter values for single-dose 1 mg/kg boluss.c. administration in normal male Sprague-Dawley rats. Parameter CmaxTerminal AUC_(0−>inf) MRT Cl/f Vz/f Compound (n) (ng/ml) t_(1/2) (h) (ng× hr/ml) (h) (ml/hr/kg) (ml/kg) WHO hGH (3) 529 (±127) 0.53 (±0.07) 759(±178)  1.29 (±0.05) 1,368 (±327)  1051 (±279)  (his)hGH (4) 680 (±167)0.61 (±0.05) 1,033 (±92)    1.30 (±0.17) 974 (±84) 853 (±91) 30KPEG-pAF35(his)hGH (4) 1,885 (±1,011) 4.85 (±0.80) 39,918 (±22,683)19.16 (±4.00)  35 (±27) 268 (±236) 30KPEG-pAF92(his)hGH (6) 663 (±277)4.51 (±0.90) 10,539 (±6,639)  15.05 (±2.07) 135 (±90) 959 (±833)30KPEG-pAF131(his)hGH (5) 497 (±187) 4.41 (±0.27) 6,978 (±2,573) 14.28(±0.92) 161 (±61) 1,039 (±449)  30KPEG-pAF134(his)hGH (3) 566 (±204)4.36 (±0.33) 7,304 (±2,494) 12.15 (±1.03) 151 (±63) 931 (±310)30KPEG-pAF143(his)hGH (5) 803 (±149) 6.02 (±1.43) 17,494 (±3,654)  18.83(±1.59)  59 (±11) 526 (±213) 30KPEG-pAF145(his)hGH (5) 634 (±256) 5.87(±0.09) 13,162 (±6,726)  17.82 (±0.56)  88 (±29) 743 (±252)

Example 91 Pharmacodynamic Studies

Hypophysectomized male Sprague-Dawley rats were obtained from CharlesRiver Laboratories. Pituitaries were surgically removed at 3-4 weeks ofage. Animals were allowed to acclimate for a period of three weeks,during which time bodyweight was monitored. Animals with a bodyweightgain of 0-8 g over a period of seven days before the start of the studywere included and randomized to treatment groups. Rats were administeredeither a bolus dose or daily dose subcutaneously. Throughout the studyrats were daily and sequentially weighed, anesthetized, bled, and dosed(when applicable). Blood was collected from the orbital sinus using aheparinized capillary tube and placed into an EDTA coated microfugetube. Plasma was isolated by centrifugation and stored at −80° C. untilanalysis. The mean (+/S.D.) plasma concentrations were plotted versustime intervals.

The peptide IGF-1 is a member of the family of somatomedins orinsulin-like growth factors. IGF-1 mediates many of the growth-promotingeffects of growth hormone. IGF-1 concentrations were measured using acompetitive binding enzyme immunoassay kit against the providedrat/mouse IGF-1 standards (Diagnosic Systems Laboratories).Hypophysectomized rats. Rats (n=5-7 per group) were given either asingle dose or daily dose subcutaneously. Animals were sequentiallyweighed, anesthetized, bled, and dosed (when applicable) daily.Bodyweight results are taken for placebo treatments, wild type hGH(hGH), His-tagged hGH ((his)_(h)GH), and hGH polypeptides comprisingp-acetyl-phenylalanine covalently-linked to 30 kDa PEG at positions 35and 92. The bodyweight gain at day 9 for 30 KPEG-pAF35(his)_(h)GHcompound was observed to be statistically different (p<0.0005) from the30 KPEG-pAF92(his)_(h)GH compound, in that greater weight gain wasobserved. The effect on circulating plasma IGF-1 levels afteradministration of a single dose of hGH polypeptides comprising anon-naturally encoded amino acid that is PEGylated, with significantdifference determined by t-test using two-tailed distribution, unpaired,equal variance.

Example 92 Human Clinical Trial of the Safety and/or Efficacy ofPEGylated hGH Comprising a Non-Naturally Encoded Amino Acid

Objective To compare the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human hGH comprising a non-naturallyencoded amino acid with one or more of the commercially available hGHproducts (including, but not limited to Humatrope™ (Eli Lilly & Co.),Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer)and Saizen/Serostim™ (Serono)).

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to hGH within threemonths of study entry; had an illness within seven days of study entry;and have significant abnormalities on the pre-study physical examinationor the clinical laboratory evaluations within 14 days of study entry.All subjects are evaluated for safety and all blood collections forpharmacokinetic analysis are collected as scheduled. All studies areperformed with institutional ethics committee approval and patientconsent.

Study Design This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). GH is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated hGH comprising a non-naturally encodedamino acid and the commercially available product chosen. The dose andfrequency of administration of the commercially available product is asinstructed in the package label. Additional dosing, dosing frequency, orother parameter as desired, using the commercially available productsmay be added to the study by including additional groups of subjects.Each dosing period is separated by a 14-day washout period. Subjects areconfined to the study center at least 12 hours prior to and 72 hoursfollowing dosing for each of the two dosing periods, but not betweendosing periods. Additional groups of subjects may be added if there areto be additional dosing, frequency, or other parameter, to be tested forthe PEGylated hGH as well. Multiple formulations of GH that are approvedfor human use may be used in this study. Humatrope™ (Eli Lilly & Co.),Nutropin™(Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer)and Saizen/Serostim™ (Serono)) are commercially available GH productsapproved for human use. The experimental formulation of hGH is thePEGylated hGH comprising a non-naturally encoded amino acid.

Blood Sampling Serial blood is drawn by direct vein puncture before andafter administration of hGH. Venous blood samples (5 mL) fordetermination of serum GH concentrations are obtained at about 30, 20,and 10 minutes prior to dosing (3 baseline samples) and at approximatelythe following times after dosing: 30 minutes and at 1, 2, 5, 8, 12, 15,18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided intotwo aliquots. All serum samples are stored at −20° C. Serum samples areshipped on dry ice. Fasting clinical laboratory tests (hematology, serumchemistry, and urinalysis) are performed immediately prior to theinitial dose on day 1, the morning of day 4, immediately prior to dosingon day 16, and the morning of day 19.

Bioanalytical Methods An ELISA kit procedure (Diagnostic SystemsLaboratory [DSL], Webster Tex.), is used for the determination of serumGH concentrations.

Safety Determinations Vital signs are recorded immediately prior to eachdosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline GH concentrations by subtracting from each of thepost-dose values the mean baseline GH concentration determined fromaveraging the GH levels from the three samples collected at 30, 20, and10 minutes before dosing. Pre-dose serum GH concentrations are notincluded in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline GHconcentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(C_(max)); time to peak serum concentration (t_(max)); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC₀₋₇₂) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t_(1/2)), computed from theelimination rate constant. The elimination rate constant is estimated bylinear regression of consecutive data points in the terminal linearregion of the log-linear concentration-time plot. The mean, standarddeviation (SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results Mean serum GH concentration-time profiles(uncorrected for baseline GH levels) in all 18 subjects after receivinga single dose of one or more of commercially available hGH products(including, but not limited to Humatrope™ (Eli Lilly & Co.), Nutropin™(Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer) andSaizen/Serostim™ (Serono)) are compared to the PEGylated hGH comprisinga non-naturally encoded amino acid at each time point measured. Allsubjects should have pre-dose baseline GH concentrations within thenormal physiologic range. Pharmacokinetic parameters are determined fromserum data corrected for pre-dose mean baseline GH concentrations andthe C_(max) and t_(max) are determined. The mean t_(max) for theclinical comparator(s) chosen (Humatrope™ (Eli Lilly & Co.), Nutropin™(Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer),Saizen/Serostim™ (Serono)) is significantly shorter than the t_(max) forthe PEGylated hGH comprising the non-naturally encoded amino acid.Terminal half-life values are significantly shorter for the commerciallyavailable hGH products tested compared with the terminal half-life forthe PEGylated hGH comprising a non-naturally encoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations; such as male or femalepatients with cancer or chronic renal failure, pediatric renal failurepatients, patients in autologous predeposit programs, or patientsscheduled for elective surgery.

In conclusion, subcutaneously administered single doses of PEGylated hGHcomprising non-naturally encoded amino acid will be safe and welltolerated by healthy male subjects. Based on a comparative incidence ofadverse events, clinical laboratory values, vital signs, and physicalexamination results, the safety profiles of the commercially availableforms of hGH and PEGylated hGH comprising non-naturally encoded aminoacid will be equivalent. The PEGylated hGH comprising non-naturallyencoded amino acid potentially provides large clinical utility topatients and health care providers.

Example 93 Comparison of Water Solubility of PEGylated hGH andNon-PEGylated hGH

The water solubility of hGH wild-type protein (WHO hGH), His-tagged hGHpolypeptide (his-hGH), or His-tagged hGH polypeptide comprisingnon-natural amino acid p-acetyl-phenylalanine covalently linked to 30kDa PEG at position 92 are obtained by determining the quantity of therespective polypeptides which can dissolve on 100 μL of water. Thequantity of PEGylated hGH is larger than the quantities for WHO hGH andhGH which shows a that PEGylation of non-natural amino acid polypeptidesincreases the water solubility.

Example 94 In Vivo Studies of Modified Therapeutically ActiveNon-Natural Amino Acid Polypeptide

Prostate cancer tumor xenografts are implanted into mice which are thenseparated into two groups. One group is treated daily with a modifiedtherapeutically active non-natural amino acid polypeptide and the othergroup is treated daily with therapeutically active natural amino acidpolypeptide. The tumor size is measured daily and the modifiedtherapeutically active non-natural amino acid polypeptide has improvedtherapeutic effectiveness compared to the therapeutically active naturalamino acid polypeptide as indicated by a decrease in tumor size for thegroup treated with the modified therapeutically active non-natural aminoacid polypeptide.

Example 95 In Vivo Studies of Modified Therapeutically ActiveNon-Natural Amino Acid Polypeptide

Prostate cancer tumor xenografts are implanted into mice which are thenseparated into two groups. One group is treated daily with a modifiedtherapeutically active non-natural amino acid polypeptide and the othergroup is treated daily with therapeutically active natural amino acidpolypeptide. The tumor size is measured daily and the modifiedtherapeutically active non-natural amino acid polypeptide has improvedtherapeutic effectiveness compared to the therapeutically active naturalamino acid polypeptide as indicated by a decrease in tumor size for thegroup treated with the modified therapeutically active non-natural aminoacid polypeptide.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of producing a polypeptide comprising at least one aminoacid having a structure of Formula (I):

the method comprising incorporating the amino acid of Formula (I) into aterminal or internal position within the polypeptide, wherein: A isoptional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkyleneor substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substitutedalkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; J is

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;each R″ is independently H, alkyl, substituted alkyl, or a protectinggroup, or when more than one R″ group is present, two R″ optionally forma heterocycloalkyl; R₁ is H, an amino protecting group, resin, aminoacid, polypeptide, or polynucleotide; and R₂ is OH, an ester protectinggroup, resin, amino acid, polypeptide, or polynucleotide; each of R₃ andR₄ is independently H, halogen, lower alkyl, or substituted lower alkyl,or R₃ and R₄ or two R₃ groups optionally form a cycloalkyl or aheterocycloalkyl; or the -A-B-J-R groups together form a bicyclic ortricyclic cycloalkyl or heterocycloalkyl comprising at least onecarbonyl group, including a dicarbonyl group, protected carbonyl group,including a protected dicarbonyl group, or masked carbonyl group,including a masked dicarbonyl group; or the -J-R group together forms amonocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising atleast one carbonyl group, including a dicarbonyl group, protectedcarbonyl group, including a protected dicarbonyl group, or maskedcarbonyl group, including a masked dicarbonyl group; with a proviso thatwhen A is phenylene and each R₃ is H, B is present; and that when A is(CH₂)₄— and each R₃ is H, B is not —NHC(O)(CH₂CH₂)—; and that when A andB are absent and each R₃ is H, R is not methyl.
 2. The method of claim1, wherein the amino acid is incorporated at a specific site into thepolypeptide using a translation system comprising: (i) a polynucleotideencoding the polypeptide, wherein the polynucleotide comprises aselector codon corresponding to the pre-designated site of incorporationof the amino acid of Formula (I), and (ii) a tRNA comprising the aminoacid, wherein the tRNA is specific to the selector codon.
 3. The methodof claim 2, wherein the translation system comprises a tRNA that isaminoacylated to the amino acid of Formula (I).
 4. The method of claim3, wherein the translation system is an in vivo translation systemcomprising a cell selected from the group consisting of a bacterialcell, archeaebacterial cell, and eukaryotic cell.
 5. The method of claim4, wherein the amino acid has a structure corresponding to Formula(III):

wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl, or substituted alkyl.
 6. The method of claim 5,wherein the amino acid is selected from the group consisting of:


7. The method of claim 4, wherein the amino acid has a structurecorresponding to Formula (VI):

wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl substituted alkyl —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl or substituted alkyl.
 8. The method of claim 7,wherein the amino acid is selected from the group consisting of:


9. The method of claim 4, wherein the amino acid has a structurecorresponding to Formula (IX):

wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl or substituted alkyl.
 10. The method of claim 9,wherein the amino acid is selected from the group consisting of:


11. The method of claim 4, wherein the -A-B-J-R groups together form abicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at leastone carbonyl group, including a dicarbonyl group, protected carbonylgroup, including a protected dicarbonyl group, or masked carbonyl group,including a masked dicarbonyl group.
 12. The method of claim 11 whereinthe amino acid is selected from the group consisting of:


13. The method of claim 4, wherein the -J-R group together forms amonocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising atleast one carbonyl group, including a dicarbonyl group, protectedcarbonyl group, including a protected dicarbonyl group, or maskedcarbonyl group, including a masked dicarbonyl group.
 14. The method ofclaim 13 wherein the amino acid is:


15. The method of claim 4, wherein the amino acid of Formula (I) has thestructure of Formula (XXX):

wherein X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene).
 16. The method of claim4, wherein the amino acid of Formula (I) has the structure of Formula(XXXIII):

wherein X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene).
 17. The method of claim4, corresponding to Formula (XXXX):

wherein: M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups; and T₃ is a bond, C(R)(R), O, or S, and R isH, halogen, alkyl substituted alkyl cycloalkyl, or substitutedcycloalkyl;
 18. The method of claim 17, corresponding to Formula(XXXXIII):


19. A method for derivatizing a polypeptide comprising an amino acid ofFormula (I), the method comprising contacting the polypeptide with areagent of Formula (XIX), wherein Formula (I) corresponds to:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower alkenylene, substituted lower alkenylene, arylene,substituted arylene, heteroarylene, substituted heteroarylene,alkarylene, substituted alkarylene, aralkylene, or substitutedaralkylene; B is optional, and when present is a linker selected fromthe group consisting of lower alkylene, substituted lower alkylene,lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,—S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substitutedalkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; J is

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;each R′ is independently H, alkyl, or substituted alkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each R₃ and R₄ is independently H,halogen, lower alkyl, or substituted lower alkyl; wherein Formula (XIX)corresponds to:[X-L]_(n)-L₁W  (XIX) wherein: each X is independently a detectablelabel, biologically active agent, or polymer; each L is a linkerindependently selected from the group consisting of alkylene,substituted alkylene, alkenylene, substituted alkenylene, —O—,—O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substitutedalkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene orsubstituted alkylene)-, —C(O)—, —C(O)-(alkylene or substitutedalkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, -(alkylene or substitutedalkylene)NR′C(O)O-(alkylene or substituted alkylene)-,—O—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(O)O-(alkylene orsubstituted alkylene)-, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)N(R′)-(alkylene or substituted alkylene)-, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; L₁ is optional, and whenpresent, is —C(R′)_(p)—NR′ —C(O)O-(alkylene or substituted alkylene)-where p is 0, 1, or 2; W is —ON(R¹)₂ or C(═O)R₂, where each R₁ isindependently H or an amino protecting group, and R₂ is H or OR′; and nis 1 to
 3. 20. The method of claim 19, wherein the amino acidcorresponds to Formula (II):


21. The method of claim 19, wherein the reagent corresponds to Formula(XXVII):


22. The method of claim 19, wherein the derivatized polypeptidecomprises at least one oxime containing amino acid having the structureof Formula (XI):

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower alkenylene, substituted lower alkenylene, arylene,substituted arylene, heteroarylene, substituted heteroarylene,alkarylene, substituted alkarylene, aralkylene, or substitutedaralkylene; B is optional, and when present is a linker selected fromthe group consisting of lower alkylene, substituted lower alkylene,lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,—S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substitutedalkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R is H, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each R₃ and R₄ is independently H, halogen, lower alkyl,or substituted lower alkyl; R₅ is H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,substituted aralkyl, —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″is independently hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substitutedaralkyl; or R₅ is L-X, where X is a detectable label, biologicallyactive agent, or polymer; and L is optional, and when present is alinker selected from the group consisting of alkylene, substitutedalkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,—S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substitutedalkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; with a proviso that when Aand B are absent, R is not methyl.
 23. The method of claim 19, whereinthe polypeptide is contacted with the reagent of Formula (XIX) inaqueous solution under mildly acidic conditions.
 24. The method of claim23, wherein the conditions are pH 2 to
 8. 25. The method of claim 19,wherein the polypeptide is contacted with the reagent of Formula (XIX)in the presence of an accelerant selected from the group consisting of:


26. The method of claim 19, wherein the amino acid of Formula (I) hasthe structure of Formula (XXX):

wherein X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene).
 27. The method of claim19, wherein the amino acid of Formula (I) has the structure of Formula(XXXIII):

wherein X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene).
 28. The method of claim19, corresponding to Formula (XXXX):

wherein: M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups; and T₃ is a bond, C(R)(R), O, or S, and R isH, halogen, alkyl substituted alkyl cycloalkyl, or substitutedcycloalkyl;
 29. The method of claim 28, corresponding to Formula(XXXXIII):